Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T05:30:27Z (GMT). No. of bitstreams: 1 Lanaro_Carolina_D.pdf: 2434106 bytes, checksum: 653633a8c42bec18d4e48b182ef0e289 (MD5) Previous issue date: 2007 / Resumo: A anemia falciforme (AF) é causada pela substituição de uma única base no códon 6 do gene da globina beta (GAG - GTG), levando à produção de hemoglobina S. Estudos recentes têm mostrado que a ativação de guanilato ciclase (GCs) em linhagens de células eritrocíticas e em cultura primária de precursores da série vermelha, causa um aumento na transcrição do gene gama globina e conseqüentemente aumento na produção de hemoglobina fetal (Hb F) (Ikuta et al., 2001). Os nucleotídeos cíclicos, AMPc e GMPc são segundos mensageiros responsáveis por vários sinais extracelulares, incluindo hormônios e neurotransmissores e provavelmente têm papel importante na anemia falciforme regulando a produção de HbF. Este trabalho teve como objetivo, primeiramente, estabelecer um sistema de cultura de células CD 34+ e posteriormente investigar a existência de uma via NO-GMPc/AMPc envolvida na produção de Hb F nos eritrócitos de pacientes com anemia falciforme e verificar se haveria aumento da expressão de GCs e de gama globina nestes mesmos pacientes. Amostras de sangue foram coletadas de voluntários sadios e pacientes com anemia falciforme. Foi utilizado cultura de células CD34+ hematopoéticas de sangue periférico, e ao longo da diferenciação eritróide (7°, 10° e 13° dia) foram coletadas células para citometria de fluxo, citospin, dosagem de GMPc e AMPc e avaliação da expressão gênica de gama globina, GCs alfa e beta subunidades, eNOS, PDE3B, CD36, CD29B e CD49. Observamos que gama globina encontra-se significativamente mais expressa nas culturas falciformes no 13° dia de diferenciação eritróide em relação ao mesmo ponto das culturas controle (2,945 ± 0,303 x 1,445 ± 0,165, respectivamente ,p=0,0095, n=6). A expressão de GCs alfa e beta subunidades, eNOS, PDE3B, CD36, CD29B e CD49 não apresentou diferenças significativas durante a diferenciação eritróide nas culturas falciformes e controles. Os níveis de GMPc nas culturas de pacientes com anemia falciforme estavam significativamente diminuídos ao longo da eritropoese in vitro em comparação com as culturas controles (dia 7: 0,2765 ± 0,0527 x 0,639 ± 0,1221 (p=0,0260); dia 10: 0,1166 ± 0,0219 x 0,268 ± 0,1181; dia 13: 0,037 ± 0,0061 x 0,093 ± 0,0236 (p=0,0152), respectivamente), enquanto o AMPc, encontrava-se elevado no dia 7 das culturas falciformes em relação às culturas controles (dia 7: 72,017 ± 14,849 x 24,987 ± 13,497 (p=0,048); dia 10: 36,3384 ± 16,708 x 8,663 ± 4,591; dia 13: 1,7138 ± 1,093 x 0,680 ± 0,370, respectivamente). O tratamento com BAY 41-2272 (ativador de GCs) em única cultura controle aumentou aproximadamente 3x os níveis de gama globina após 72 h de incubação. Nossos resultados sugerem que as células tronco hematopoéticas dos pacientes com AF são diferentes às dos controles, uma vez que ambas, sob as mesmas condições in vitro expressaram níveis significativamente diferentes de gama globina. Um cross talk entre o GMPc e o AMPc poderia regular a expressão de gama globina ao longo da diferenciação eritróide. Estes dados resultam no primeiro trabalho a demonstrar a expressão basal de gama globina ao longo da diferenciação eritróide de pacientes com anemia falciforme. Desta maneira, o esclarecimento desta via pode contribuir com entendimento da fisiopatologia da anemia falciforme e identificar novos alvos para a regulação farmacológica de hemoglobina fetal / Abstract: Sickle cell disease (SCD) is caused by the substitution of a single base in codon 6 of the beta globin gene (GAG-GTG), causing the production of S hemoglobin. Recent studies have demonstrated that the activation of soluble guanylate cyclase (sGC) in erythrocytic cell lines and in primary erythroid precursor culture causes an increase in the transcription of the gamma globin gene and, consequently, an augmented production of fetal hemoglobin (HbF) (Ikuta et al., 2001). The cyclic nucleotides, cAMP and cGMP, are second messengers that are responsible for mediating the signaling of various extracellular signals, including those of hormones and neurotransmitters and, probably, have an important role in SCD by regulating the production of HbF. This study aimed to investigate the existence of a NO-cAMP/cAMP pathway involved in the production of HbF in the erythroid cells of patients with SCD and to determine whether there is an increase in the expression of sGC and of gamma globin in these same patients. Blood samples were collected from healthy volunteers and SCD patients. Peripheral blood hematopoietic CD34+ cell culture was used for the investigation; cells were differentiated into erythroid lineage and cells collected (7th, 10th and 13th day) for flow cytometry, citospin, measurement of cGMP/cAMP and evaluation of the gene expression of gamma globin, the sGC alpha and beta subunits, eNOS, PDE3B, CD36, CD29B and CD49. We demonstrate that gamma globin was significantly more expressed in the cultures of SCD cells on the 13th day of erythroid differentiation compared to the same time point in control cells (2.95 ± 0.30 vs. 1.45 ± 0.17, respectively, p=0.0095, n=6). The expression of sGC alpha and beta subunits, eNOS, PDE3B, CD36, CD29B and CD49 did not present significant differences during erythroid differentiation in control or SCD cell cultures. Levels of cGMP were significantly decreased during erythropoiesis in the SCD cultures compared to control cultures (day 7: 0.277 ± 0.053 vs. 0.639 ± 0.122 (p=0.026); day 10: 0.117 ± 0.022 vs. 0.268 ± 0,118; day 13: 0.037 ± 0.006 vs. 0.093 ± 0.024 (p=0.015), respectively). In contrast, levels of cAMP were significantly increased in the SCD cultures compared to the control cultures (day 7: 72.02 ± 14.85 vs. 24.99 ± 13.50 (p=0.048); day 10: 36.34 ± 16.71 vs. 8.66 ± 4.59; day 13: 1.74 ± 1.09 vs. 0.680 ± 0.370, respectively). Treatment with BAY 41-2272 (sGC inducer) in a single control cell culture increased the levels of gamma globin by approximately 3 times after 72h of incubation. Results suggest that the hematopoietic stem cells of SCD patients are significantly and constitutively different to those of controls, since under the same in vitro culture conditions, differentiation of the two cell types results in consistently significant different levels of gamma globin. A cross talk between cGMP and cAMP may regulate the expression of gamma globin during erythroid differentiation in patients with SCD. Thus, further investigation of this pathway may contribute to the understanding of the physiopathology of the disease and identify new targets for the pharmacological regulation of HbF production / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

INTRODUÇÃO: Embora todos os indivíduos com anemia falciforme (AF) apresentem o mesmo defeito molecular nos genes da beta-globina, existe uma considerável variabilidade fenotípica entre eles. A síntese continuada da hemoglobina fetal (HbF) é o mais potente modificador da morbimortalidade da AF. A HbF diminui a polimerização da desoxi-HbS, reduzindo a intensidade da anemia hemolítica crônica e dos fenômenos vaso-oclusivos, consequentemente, as complicações sistêmicas da AF. Há uma grande variação na taxa de síntese de HbF (1% a 30%) em indivíduos com AF. Vários estudos demonstraram que o tratamento com hidroxiureia (HU) diminui a morbimortalidade desta hemoglobinopatia, principalmente, por estimular a síntese de HbF. Na dose máxima tolerada (DMT), a HU geralmente aumenta os níveis de HbF entre 10% e 40%. Contudo, há uma grande variabilidade de resposta, a DMT é muito variável, cerca de 25% dos portadores de AF não respondem e não há preditores de resposta definidos à HU. Estudos polimorfismos de nucleotídeo único (SNPs) demonstraram a influência de modificadores epigenéticos na regulação da expressão da HbF. Estes elementos são, principalmente, o oncogene BCL11A, a região intergênica HMIP e o polimorfismo Xmn1-HBG2. Estes 3 quantitative trait loci (QTLs) detêm 20% a 50% do controle da expressão dos genes HBG (genes envolvidos na síntese da HbF). OBJETIVOS: Avaliar o comportamento epidemiológico e a associação dos SNPs Xmn1- HBG2; BCL11A rs7482144, rs4671393 e rs11886868; HMIP-2 rs9399137 e rs9402686 com a expressão da HbF e com o comportamento dos parâmetros hematimétricos em portadores de AF tratados com HU. PACIENTES E MÉTODOS: Neste estudo pioneiro de coorte prospectivo foram incluídos sequencialmente indivíduos com AF, em uso regular de HU por pelo menos 6 meses, que não receberam transfusão sanguínea 3 meses antes de ingressar no estudo e que não faziam uso de drogas que estimulassem a síntese de HbF. Foram coletados 4 ml de sangue venoso periférico para extração do DNA genômico. A genotipagem dos polimorfismos foi realizada por meio da reação da cadeia de polimerase em tempo real. RESULTADOS: Foram avaliados 121 pacientes, entre 1 ano e 9 meses-54 anos (19 ± 14) anos idade, que estavam recebendo doses regulares de HU entre 8,6-42,8 (23 ± 7,6) mg/kg/dia, durante 6- 254 (102± 67) meses. Não encontramos correlação entre a contagem de leucócitos, de neutrófilos e de reticulócitos; hemoglobina total; volume corpuscular médio e a concentração de hemoglobina corpuscular média, com os valores basais da HbF. A HbF basal (r=0,40; P < 0,001), a hemoglobina total basal (r=0,26; P = 0,005) e o tempo de exposição (r = -0,30; P = 0,001) foram associadas significativamente com maiores taxas de HbF ao final do estudo. Não houve correlação dos polimorfismos com os parâmetros hematimétricos, com o tempo de exposição e com a DMT de HU. Os SNPs HMIP-2 rs9399137 e rs9402686 foram responsáveis por 5,7% e 8,4% do total de variação da HbF basal (P= 0,01 e P=0,002). Não houve correlação, porém, entre os demais polimorfismos com a variação dos níveis basais de HbF. Os SNPs BCL11A rs1427407, rs4671393 e rs11886868 foram responsáveis, respectivamente, por uma variação de 7,6%, 4,5% e 4,3% nos níveis de HbF final (P=0,017; P=0,025 e P=0,029). Ainda, houve uma associação do rs1427407 (B = 0,29; P = 0,035) e do rs4671393 (B = 0,28; P = 0,036) em relação aos valores do delta HbF (variação da HbF final menos a HbF inicial). CONCLUSÃO: Estes dados sugerem que os indivíduos com AF com SNP BCL11A rs1427407 respondem mais favoravelmente ao tratamento com HU, no incremento dos níveis de HbF. São necessários estudos com populações maiores para validarmos estes achados. / INTRODUCTION: Although all individuals with sickle cell anemia (SCA) have the same molecular defect in the beta-globin genes, considerable phenotypic variability exists between them. Continued synthesis of fetal hemoglobin (HbF) is the most powerful SCA morbimortality modifier. HbF decreases the polymerization of deoxy-Hb, reducing the intensity of chronic hemolytic anemia and vaso-occlusive phenomena, and consequently systemic complications of SCA. There is a large variation in the HbF synthesis rate (1 to 30%) in patients with SCA. Several studies show that treatment with hydroxyurea (HU) decreases the morbimortality of this hemoglobinopathy, mainly by stimulating HbF synthesis. At the maximum tolerated dose (MTD), HU generally increases HbF levels from 10 to 40%. However, there is great variability in response as the MTD is highly variable, about 25% of SCA patients do not respond and there are no response predictors set for HU. Single nucleotide polymorphism studies (SNPs) demonstrate the influence of epigenetic modifiers in the regulation of HbF expression. These elements are, mainly, the BCL11A oncogene, the HMIP intergenic region and the Xmn1-HBG2 polymorphism. These 3 quantitative trait loci (QTLs) hold 20 to 50% of HBG gene expression control (genes involved in HbF synthesis). OBJECTIVE: To study the epidemiological behaviors and the association of SNPs Xmn1- HBG2; BCL11A rs7482144, rs4671393 and rs11886868; HMIP-2 rs9399137 and rs9402686 with HbF expression and with the behavior of hematimetric parameters in SCA patients treated with HU. PATIENTS AND METHODS: In this pioneering prospective cohort study, we included SCA patients, in regular treatment with HU at least for 6 months, who had not received blood transfusions in the 3 months prior to entering in the study and who didn‘t use drugs that stimulate HbF synthesis. We collected 4 ml of venous blood to proceed with the genomic DNA extraction. The polymorphism genotyping was done by real-time polymerase chain reaction. RESULTS: We evaluated 121 individuals with SCA aged between 1 year 9 months and 54 years (19 ± 14) who were receiving HU doses between 8.6 and 42.8 (23 ± 7.6) mg/kg/day for 6 to 254 (102 ± 67) months. No correlation was found between total leukocyte, neutrophils and reticulocytes counts; total hemoglobin; mean corpuscular volume and the concentration of mean corpuscular hemoglobin with baseline values of HbF. Basal HbF (r = 0.40; P <0.001), total baseline hemoglobin (r = 0.26; P = 0.005) and exposure time to HU (r = -0.30; P = 0.001) were significantly associated with higher HbF rates at the end of the study. There was no correlation of polymorphisms with the hematological parameters, exposure time and the MTD of HU. The SNPs HMIP-2, rs9399137 and rs9402686 accounted for 5.7% and 8.4% of the total variation of baseline HbF (P = 0.01 and P = 0.002). There was no correlation, however, between the other polymorphisms and variation in baseline HbF levels. The SNPs BCL11, rs1427407, rs4671393 and rs11886868 were responsible, respectively, for a variation of 7.6%, 4.5% and 4.3% in the final HbF levels (P = 0.017, P = 0.025 and P = 0.029). Still, there was an association of rs1427407 (B = 0.29; P = 0.035) and rs4671393 (B = 0.28; P = 0.036) in relation to delta HbF values (final minus initial HbF variation). CONCLUSIONS: These data suggest that individuals with SCA who have SNP rs1427407 BCL11A respond more favorably to HU treatment, with increased HbF levels. Studies with larger populations are necessary to validate these findings.

Bakgrund: Glykerat hemoglobin i blod (B-HbA1c) speglar den genomsnittliga blodsocker-nivån de senaste 8 till 12 veckorna. B-HbA1c används för att följa behandlingen vid diabetes mellitus. B-HbA1c kan mätas på olika sätt och mätresultaten kan ibland skilja sig åt. I detta arbete har en ny immunkemisk metod, HbA1c Advanced (Beckman Coulter, USA) (AU-metoden) utvärderats.   Metod: Med patientprover och kontroller, dubbelprover, beräknades imprecision och bias samt korrelation med laboratoriets ordinarie jonbyteskromatografiska metod, Tosoh G11 (G11-metoden).   Resultat: Imprecision inomserie blev &lt;3 %, total imprecision 5 %, och bias var -2,8 till -5,3 %. AU-metoden gav i medeltal 2,5 mmol/mol (3,9 %) lägre värde än G11-metoden med en korrelationskoefficient på 0,9967. AU-metoden mätte korrekt de vanligaste hemoglobin-varianterna men inte en patologisk ökning av fraktionen HbF.      Slutsats: AU-metodens precision och bias uppfyller nationella kvalitetsmål och korrelationen med laboratoriets ordinarie metod är god för såväl prover utan som med de vanligare hemo-globinvarianterna. Som förväntat för immunkemiska metoder ger AU-metoden för lågt värde vid uttalad ökning av HbF.   AU-metoden fungerar så väl som en immunkemisk metod kan göra och bedöms vara lämplig för kliniskt bruk för att mäta B-HbA1c. / Background: Glycated hemoglobinin blood (B-HbA1c) reflects the average blood sugar level over the last 8 to 12 weeks. B-HbA1c is used to monitor the treatment of diabetes mellitus. B-HbA1c can be measured in different ways with eventual results that sometimes differ from each other. During this project, a new immunochemical method, HbA1c Advanced (Beckman Coulter, USA) (AU method) was evaluated.   Methods: With patient samples, precision and bias were calculated as well as correlation with the laboratory's ordinary ion exchange chromatographic method, Tosoh G11 (G11 method).    Results: Imprecision in-series was &lt;3 %, total imprecision was 5 %, and bias was -2.8 to -5.3  %. On average, the AU method gave values that were lower than the G11 method (2.5 mmol/mol (3.9 %) with a correlation coefficient of 0.9967. The AU method correctly measured the most common hemoglobin variants but not a pathological increase in the HbF fraction.   Conclusion: The precision and bias of the AU method fulfill the national quality objectives and the correlation with the laboratory's ordinary method, the G11 method is good for samples both with and without the most common hemoglobin variants. As expected for immunochemical methods, the AU method gives too low a value for samples with a pronounced increase in HbF.    In conclusion, the AU method fulfills the quality goals as is expected for an immunochemical method for measuring B-hbA1c and is concidered appropriate to use in clincical work.

Sickle cell disease is a disabling chronic autosomal recessive blood disease characterized by abnormal hemoglobin, pain crises, and frequent emergency department visits. Adherence to hydroxyurea therapy has been shown to improve these patient outcomes. Guided by the theory of comfort, the purpose of this project was to determine if an educational intervention would increase adherence to hydroxyurea therapy in pediatric patients between 2 and 17 years of age recruited from an urban university hospital hematology clinic. The RE-AIM model was used to support the translation of evidence and the change process. An educational video produced by AFLAC was viewed by patients' parents 4 weeks after enrollment into this pretest/posttest design project. A total of 22 African-American parent participants completed the 8-item Morisky Medication Adherence Scale at baseline and again at 8 weeks to assess hydroxyurea adherence. The Short Test of Functional Health Literacy in Adults tool was used to assess parents' health learning needs; all parents met the adequate literacy level at baseline. Using t test statistics, no statistically significant differences were found pretest to posttest on the Morisky Medication Adherence Scale scores, mean corpuscular hemoglobin, and fetal hemoglobin percentages. Wilcoxon Signed Rank tests showed no significant differences in emergency room visits nor number of pain crisis. Although no significant changes emerged in short-term hematologic findings, emergency room visits, and pain crises, social change in the health care setting was promoted by confirming parents were able to understand education and a high level of hydroxyurea adherence was maintained; literature indicated that long-term adherence to hydroxyurea limits severe attacks.

Τα αυξημένα επίπεδα εμβρυϊκής αιμοσφαιρίνης (HbF) μετριάζουν την βαρύτητα των διαταραχών που αφορούν στην β-σφαιρίνη, δηλαδή τη δρεπανοκυτταρική αναιμία (SCD) και την β-μεσογειακή αναιμία, που αποτελούν σημαντικές αιτίες παγκόσμιας νοσηρότητας και θνησιμότητας. Γι’ αυτό το λόγο είναι μακροχρόνιο το ενδιαφέρον για την ανάπτυξη θεραπευτικών προσεγγίσεων για την επαγωγή της παραγωγής HbF. Η αναζήτηση μορίων που ρυθμίζουν την μετάβαση από την έκφραση της εμβρυϊκής (HbF) στην έκφραση της αιμοσφαιρίνης των ενηλίκων (HbA) και που συντελούν στην διατήρηση της αποσιώπησης ή αντίθετα στην ενεργοποίηση της έκφρασης της HbF στους ανθρώπους αποτελεί πολυετές αντικείμενο έρευνας με σκοπό την στόχευση αυτών των παραγόντων για την επαγωγή της HbF (Sankaran et al. 2011). Έτσι, εκτός από τα cis-ρυθμιστικά στοιχεία, έχουν εντοπιστεί και trans-ρυθμιστικά στοιχεία, τα οποία αποτελούν κυρίως μεταγραφικούς παράγοντες. Όμως υπάρχουν και γονιδιακοί τόποι εκτός του β-συμπλέγματος που φαίνεται να επιδούν στην ρύθμιση της έκφρασης των γονιδίων του β-γονιδιακού τόπου. Τέτοιοι είναι οι τόποι που συνδέονται με «ποσοτικά γνωρίσματα» (Quantitative trait loci-QTL). Η χρωμοσωμική περιοχή 6q23 έχει σε διάφορες μελέτες προσδιοριστεί ως QTL, που συνδέεται με την μεταβολή των επιπέδων της HbF σε ασθενείς με SCD. (Close et al. 2004, Thein et al. 2007, Wyszynski et al. 2004). Ο σκοπός της παρούσας μελέτης είναι διττός: A. Ο εντοπισμός μονονουκλεοτιδικών πολυμορφισμών (SNP) εντός των γονιδίων MAP3K5 και PDE7B του QTL στην 6q23 χρωμοσωμική περιοχή, που να σχετίζονται με αυξημένα επίπεδα HbF. B. Η αξιολόγηση των SNP αυτών ως φαρμακογονιδιωματικών δεικτών, που να σχετίζονται με την μεταβλητότητα των επιπέδων της HbF ως απόκριση στη θεραπεία με HU. / Hemoglobinopathies, particularly β-thalassemia and sickle cell disease (SCD), are major health problems, in which quantitative or qualitative defects in hemoglobin production occur, respectively. Under normal circumstances, different types of hemoglobin (Hb) are produced during embryonic, fetal, and adult life. At birth, fetal hemoglobin (HbF), in particular, composes 80–90% of the total hemoglobin synthesized, but it gradually decreases to approximately 1% by 10 months in infancy as its synthesis is restricted to a small subset of erythrocytes termed ‘F cells’ [Patrinos&Grosveld, 2008]. The first studies searching for regulators of HbF expression were conducted on individuals with heterocellular hereditary persistence of HbF (HPFH) – i.e. increase of HbF levels unevenly distributed among ‘F cells’ – and suggested the absence of linkage between the determinant of the HbF levels and the β-globin gene cluster, back then named “non-α globin cluster” [Gianni et al., 1983]. Later, while seeking for genetic elements associated with elevated HbF levels in healthy adults, several cis-acting variants on the β-globin gene complex were unraveled, including the XmnI-Gγ (HBG2) gene promoter polymorphism [Gilman et al., 1985]. Ιn addition, variants unlinked to the β-locus (trans-acting), such as quantitative trait loci (QTLs) on Xp22 [Dover et al., 1992] and 6q23 [Craig et al., 1996] became known soon after. Initially, a study on an extensive, inbred kindred of Asian Indian origin with heterocellular HPFH revealed that a key locus controlling HPFH resides on chromosome 6q, which was fine-mapped to 6q22.3–23.1 [Craig et al., 1996]. Among the first positional candidate genes in the 6q23 region, assumed to possibly explain this QTL, were the MYB proto-oncogene and the eukaryotic release factor-similar HBS1L, as well as the mitogen-activated protein kinase kinase kinase 5 (MAP3K5) [Game et al., 2000]. In addition, genes within this region are associated with response to hydroxyurea (HU) treatment based on elevated HbF levels, in SCD patients; however, the mechanism by which this chromosome 6q22-23 QTL influences HbF levels in the context of HU treatment remains unknown [Ma et al., 2007] and very few, if any, studies have addressed this question. In continuing the global effort of scrutinizing the 6q23 region for variants accounting for the modulation of HbF production, we investigated a possible association of SNPs residing within the MAP3K5 and PDE7B genes with elevated HbF levels in β-thalassemia intermediate or major patients and normal (non-thalassemic) individuals. We also examined a cohort of 38 heterozygous SCD/β-thalassemia patients who had undergone HU therapy, in order to clarify whether there is a correlation of these SNPs with HU treatment response in patients of Hellenic origin.

L’anémie falciforme est une maladie monogénique causée par une mutation dans le locus de la β-globine. Malgré le fait que l’anémie falciforme soit une maladie monogénique, cette maladie présente une grande hétérogénéité clinique. On présume que des facteurs environnementaux et génétiques contribuent à cette hétérogénéité. Il a été observé qu’un haut taux d’hémoglobine fœtale (HbF) diminuait la sévérité et la mortalité des patients atteints de l’anémie falciforme. Le but de mon projet était d’identifier des variations génétiques modifiant la sévérité clinique de l’anémie falciforme. Dans un premier temps, nous avons effectué la cartographie-fine de trois régions précédemment associées avec le taux d’hémoglobine fœtale. Nous avons ensuite effectué des études d’association pan-génomiques avec deux complications cliniques de l’anémie falciforme ainsi qu’avec le taux d’hémoglobine fœtale. Hormis les régions déjà identifiées comme étant associées au taux d’hémoglobine fœtale, aucun locus n’a atteint le niveau significatif de la puce de génotypage. Pour identifier des groupes de gènes modérément associés au taux d’hémoglobine fœtale qui seraient impliqués dans de mêmes voies biologiques, nous avons effectué une étude des processus biologiques. Finalement, nous avons effectué l’analyse de 19 exomes de patients Jamaïcains ayant des complications cliniques mineures de l’anémie falciforme. Compte tenu de la taille des cohortes de réplication disponibles, nous n’avons pas les moyens de valider statistiquement les variations identifiées par notre étude. Cependant, nos résultats fournissent de bons gènes candidats pour des études fonctionnelles et pour les réplications futures. Nos résultats suggèrent aussi que le β-hydroxybutyrate en concentration endogène pourraient influencer le taux d’hémoglobine fœtale. De plus, nous montrons que la cartographie-fine des régions associées par des études pan-génomiques peut identifier des signaux d’association additionnels et augmenter la variation héritable expliquée par cette région. / Sickle cell disease is a monogenic disease caused by a mutation in the β-globin locus. Although it is a monogenic disease, it shows a high clinical heterogeneity. Environmental and genetic factors are thought to play a role in this heterogeneity. It has been observed that a high fetal hemoglobin (HbF) levels correlates with a diminution of the severity and mortality of patients with sickle cell disease. The goal of my project was to identify genetic modifiers of the clinical severity of sickle cell disease. First, I performed the fine-mapping of three regions previously associated with HbF levels. Second, I performed genome-wide association studies with two clinical complications of sickle cell disease as well as with HbF levels. Since no new loci reached array-wide significance for HbF levels, I performed a pathway analysis to identify additional HbF loci of smaller effect size that might implicate shared biological processes. Finally, I performed the analysis of 19 whole exomes from Jamaican sickle cell disease patients with very mild complications. In conclusion, given the sample size of the replication cohorts available, we do not currently have the means to statistically validate the association signals. However, these results provide good candidate genes for functional studies and for future replication. Our results also suggest that β-hydroxybutyrate in endogenous levels could influence HbF levels. Furthermore, we show that fine-mapping the loci associated in genome-wide association studies can identify additional signals and increase the explained heritable variation.

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.