La place des gènes non globine dans la modulation du syndrome drépanocytaire

For the WHO, UNESCO and the UN, sickle cell disease ranks fourth in their global public health priorities (behind cancer, the HIV virus and malaria). In Tunisia sickle cell disease is the 2nd hemoglobinopathy after -thalassemia, thus representing a real public health problem. The average frequency of sickle cell trait in our country is 1.89%. Sickle cell disease is a historical example of molecular monomorphism. The fact that the causative mutation is always the same while the severity of the disease is variable, suggests a multifactorial genetic modulation. We chose to search, by the approach of candidate genes, for certain modifying genes which could explain certain phenotypic characteristics of the disease. The aim of the thesis presented is to study the genetic predisposition of patients with sickle cell disease to modulate the severity of the disease in order to improve the life expectancy, morbidity and quality of life of patients. For this, polymorphisms affecting candidate genes have been the subject of molecular exploration in order to look for possible phenotype/genotype correlations. The first chapter concerned uridine diphosphoglucuronosyltransferase (UGT1A1) involved in the conjugation of bilirubin, a pigment resulting from the degradation of heme. We targeted a functional polymorphism, it is an insertion of a base pair at the level of the TATA box of the promoter which causes a decrease in enzymatic activity and consequently hyperbilirubinemia. This hyperbilirubinemia is a risk factor for the development of pigmentary cholelithiasis which is a frequent complication in sickle cell patients.The second chapter concerned genes coding for chemokines such as RANTES and its receptor DARC which are involved in the polymerization of red blood cells and consequently the occurrence of vaso-occlusive crises. CCL2 and its CCR2 receptor involved in the pro-inflammatory context of sickle cell disease. The third chapter focuses on the study of the association between genetic polymorphisms and the level of hemoglobin F. The study focused on a BCL11A gene considered as a repressor of the expression of g-globin genes in erythroblasts and also to the intergenic part HBS1L-MYP (HMIP) involved in the variation of HbF levels. Our results showed that genotypes having the allele (TA)7 in the homozygous or heterozygous state are associated with hyperbilirubinemia and the formation of gallstones. At the allelic level, (TA)7 present a risk factor for lithogenesis in pediatric and adult sickle cell patients as well as in patients without sickle cell disease but presenting with lithiasis. The (TA)8 variant is significantly associated with lithogenesis in pediatric sickle cell patients as well as in non-sickle cell patients the second chapter, no significant association was found between the chemokines: RANTES, DARC and CCR2 and the complications studied. Furthermore, our results showed a significant association between the CCL2-2518A/G polymorphism and the occurrence of vaso-occlusive crises and infections in sickle cell patients. It should be noted that this is a new association never previously described in the literature. In the third chapter, our results showed a significant association between rs 11886868 and rs 4671393 of the BCL11A gene and the variation in HbF levels in sickle cell patients. Association not found for rs 4895441 and rs 9399137 of the HMIP intergenic part. In conclusion, our results agree with the literature for some studied polymorphisms and not for others. The thesis presented draws the attention of clinicians to a few genetic variants that appear to be genetic markers for certain complications of sickle cell disease and therefore to find solutions that could mitigate the complications of the disease in patients with sickle cell disease.Pour l’OMS, l’UNESCO et l’ONU, la drépanocytose occupe le quatrième rang dans leurs priorités de santé publique mondiale (derrière le cancer, le virus du VIH et le paludisme). En Tunisie la drépanocytose est la 2ème hémoglobinopathie après la -thalassémie, représentant ainsi un vrai problème de santé publique. La fréquence moyenne du trait drépanocytaire dans notre pays est de 1.89%. La drépanocytose constitue un exemple historique de monomorphisme moléculaire. Le fait que la mutation causale soit toujours la même alors que la gravité de la maladie est variable, suggère une modulation génétique multifactorielle. Nous avons choisi de rechercher, par l'approche de gènes candidats, certains gènes modificateurs qui pourraient expliquer certaines caractéristiques phénotypiques de la maladie. Le but de la thèse présentée est l’étude de la prédisposition génétique des patients atteints de la drépanocytose à moduler la gravité de la maladie afin d’améliorer l’espérance de vie, la morbidité et la qualité de vie des patients. Pour cela des polymorphismes touchant des gènes candidats ont fait l’objet d’exploration moléculaire afin de chercher d’éventuelle corrélations phénotype/génotype. Le premier chapitre a concerné l’uridine diphosphoglucuronosyltransférase (UGT1A1) impliqué dans la conjugaison de la bilirubine, un pigment résultant de la dégradation de l’hème. On a ciblé un polymorphisme fonctionnel, il s’agit d’une insertion d’une paire de base au niveau de la TATA box du promoteur qui entraine une diminution de l’activité enzymatique et par conséquent une hyperbilirubinémie. Cette hyperbilirubinémie est un facteur de risque pour le développement de lithiase biliaire pigmentaire qui est une complication fréquente chez les drépanocytaires. Le deuxième chapitre a concerné des gènes codant pour des chimiokines comme RANTES et son récepteur DARC qui sont impliqués dans la polymérisation des globules rouges et par conséquent la survenue des crises vaso-occlusives. CCL2 et son récepteur CCR2 impliquées dans le contexte pro inflammatoire de la drépanocytose. Le troisième chapitre s’intéresse à l’étude d’association entre polymorphismes génétiques et le taux de l’hémoglobine F. L’étude s’est orientée vers un gène BCL11A considéré comme répresseur de l’expression des gènes g-globine dans les érythroblastes et aussi à la partie intergénique HBS1L-MYP (HMIP) impliquée dans la variation des taux de l’HbF. Nos résultats ont montré que les génotypes ayant l’allèle (TA)7 à l’état homozygote ou hétérozygote sont associés à une hyperbilirubinémie et à la formation des calculs biliaires. Au niveau allélique, (TA)7 présentent un facteur de risque pour la lithogénèse chez les drépanocytaires à l’âge pédiatrique et adulte ainsi que chez des patients non drépanocytaires mais présentant des lithiases. Le variant (TA)8 est associé significativement à la lithogénèse chez les drépanocytaires pédiatriques ainsi que chez les patients non drépanocytaires. Concernant le deuxième chapitre, aucune association significative n’a été retrouvée entre les chimiokines: RANTES, DARC et CCR2 et les complications étudiées. Par ailleurs, nos résultats ont montré une association significative entre le polymorphisme CCL2-2518A/G et la survenue des crises vaso-occlusives et les infections chez les drépanocytaires. Il faut noter qu’il s’agit d’une nouvelle association jamais décrite auparavant dans la littérature. Dans le troisième chapitre, nos résultats ont montré une association significative entre les rs 11886868 et rs 4671393 du gène BCL11A et la variation du taux de l’HbF chez les drépanocytaires. Association non trouvée pour les rs 4895441 et rs 9399137 de la partie intérgénique HMIP. En conclusion, nos résultats concordent avec la littérature pour certains polymorphismes étudiés et pas pour d’autres. La thèse présentée attire l’attention des cliniciens à quelques variants génétiques qui semblent être des marqueurs génétiques pour certaines complications de la drépanocytose et par conséquent à trouver des solutions qui pourraient atténuer les complications de la maladie chez les patients drépanocytaires

Rectal stromal tumor with an exceptional liver and bone metastatic locations

Gastrointestinal stromal tumours (GIST) are rare mesenchymal tumours which represent 1% to 3% of gastrointestinal neoplasm. Rectal location of GIST is extremely rare reaching 5% of GIST and only 0.1% of rectal tumours. They usually metastases to the liver (65%) and exceptionally to the bone (3%). We reported a case of rectal stromal tumour with an exceptional metastasis located in the rib. A 40-year-old man who presented with pelvic pain, associated with rectal syndrome, rectal bleeding and subocclusive episodes. Physical examination objectified a tough, budding rectal mass, with a smooth wall, localized 3cm above of anal margin. A Thoraco-abdominal computed tomography showed a large heterogeneous tissue mass, taking the whole pelvis, coming from the right-side wall of the rectum of 17.3 x 14cm. It was associated with liver and bone secondary locations. Biopsies confirmed the secondary locations of an intermediate risk GIST. Immunohistochemical study showed an overexpression of c-kit protein (CD117) and Dog1. Imatinib was prescribed to reduce the tumour size. Stromal metastatic rectal tumours in bone level are extremely rare conditions. The diagnosis is confirmed by histological examination with immune histochemical analysis. The prognosis remains poor in metastatic forms but it has been improved since the introduction of Imatinib

Implication of genetic variation at the promoter and exon1 of UGT1A1 in occurrence of cholelithiasis in Tunisia.

International audienceBilirubin is conjugated with glucoronic acid in the liver by UDP-glucuronosyltransferase 1A1 (UGT1A1). Polymorphisms at the promoter region or exon1 of UGT1A1 gene result in unconjugated hyperbilirubinemia and could be at the origin of gallstone formation. The purpose of this study is to determine whether polymorphisms in the promoter area and exon 1 of UGT1A1 can be considered as a risk factor for lithogenesis. Our study involved 76 patients with cholelithiasis as well as 141 unaffected subjects. For each subject an analysis of the bilirubin parameters was performed. We screened genetic variation in the promoter and exon1 UGT1A1 namely the A (TA) nTAA and the six following SNPs: 44T>G, 101C>A, 115C>G, 145C>T, 211G>A and 222 C>A by PCR/sequencing. Our findings show that subjects with (TA)(7) or (TA)(8) variant in their genotypes are associated with high bilirubin level. Furthermore, the comparison between patients and controls according to A(TA)nTAA variation demonstrated that (TA)(6)/(TA)(7) and (TA)(7)/(TA)(7) genotype and (TA)(7) and (TA)(8) alleles were significantly associated with an increased risk of gallstone diseases p=0.0017, p= 6.1 10(-6), p=1.5 10(-6) and p=0.025 respectively. However, polymorphisms in exon1 were normal in all studied subjects except for the 211G>A which appears to be associated with a protective effect p=7.910(-9); OR=0.03, CI95% (0.001-0.158)

rs11886868 and rs4671393 of BCL11A associated with HbF level variation and modulate clinical events among sickle cell anemia patients

International audienceAims: Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia (SCA) by inhibiting deoxy sickle hemoglobin (HbS) polymerization. HbF genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Herein, we aimed to determine whether two functional polymorphisms of BCL11A are implicated in the variation of HbF and clinical events in SCA Tunisian patients. Material and methods: The studied population consisted of 148 SCA patients with SS phenotype. The group of patients was divided into two subgroups according to the threshold point of %HbF which is 15%. Genotyping of rs11886868 and rs4671393 was performed using PCR/Sequencing. To test for trait association with the candidate SNPs, genotype and allele frequencies between `group who had %HbF 15' (controls) were compared using Pearson's chi-square test (compare 2, version 1.02). The association of each genotype and the combined genotype with complications was performed by logistic regression test. Results: Our findings showed that the majority of patients carried genotype CT of rs11886868 and genotypes AG and GG of rs4671393 present HbF level < 15%. RR = 0.08, RR = 0.176, and RR = 0.189, respectively. The results showed a significant association between the alleles T of rs11886868 and G of rs4671393 and %HbF < 15% with P = 0.016; RR = 0.39 and P = 8.9 x 10(-3): RR = 0.567, respectively. Interestingly, the C allele of the rs11886868 and the A allele of the rs46713939 were associated with an ameliorated phenotype in patient's SCA. The combination of the genotypes GG and CT explains more phenotypic variance than the sum of the two BCL11A SNPs taken individually

Genetic link with cholelithiasis among pediatric SCA Tunisian patients: Examples of UGT1A1, SLCO1A2 and SLCO1B1

International audienceAims and background: Hyperbilirubinemia is often observed in chronic hemolysis and results in the formation of pigment cholelithiasis that could be increased by the presence of defected enzymes involved in the bilirubin metabolism. Indeed, this is the first report that interested in the study of polymorphisms in genes encoded for enzymes involved in the bilirubin metabolism: rs 4149056 of SLCO1B1 and rs4149000 of SLCO1A2 in combination with rs8175347 and rs887829 of UGT1A1 in order to find a correlation between the polymorphisms studied and the presence of gallstones in a population of sickle cell anemia (SCA) pediatric Tunisians. Material and methods: Our study involved 102 unrelated Tunisian subjects. All SCA patients are children (less than 16 years old) and were characterized by hyperbilirubinemia and 52 of them have cholelithiasis. The polymorphisms of the candidate genes were analyzed for all subjects by PCR/sequencing. Genotype and allele frequencies between cases and controls were compared using Pearson's chi-square test with a significance threshold of P < 0.05 (compare 2, version 1.02). Results: The novelty of this report is that children carrying the combined genotype of the rs studied: (TA7TA7)/TT/TC/GA have a higher risk to develop gallstones (P = 0.0027, RR = 18.27 (20.0061-915.28)). Conclusion: Altogether our data provide the implication of UGT1A1 and SLCO1A2 in sickle cell anemia-related cholelithiasis

Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family

Pyruvate Kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The disease has been studied in several ethnic groups. However, it is yet an unknown pathology in Tunisia. We report here, the phenotypic and molecular investigation of PK deficiency in a Tunisian family. This study was carried out on two Tunisian brothers and members of their family. Hematological, biochemical analysis and erythrocyte PK activity were performed. The molecular characterization was carried out by gene sequencing technique. The first patient died few hours after birth by hydrops fetalis, the second one presented with neonatal jaundice and severe anemia necessitating urgent blood transfusion. This severe clinical picture is the result of a homozygous mutation of PKLR gene at exon 8 (c.1079G>A; p.Cys360Tyr). Certainly, this research allowed us to correlate the clinical phenotype severity with the identified mutation. Moreover, this will help in understanding the etiology of unknown anemia in our country

Association of rs1319868, rs1567811 and rs8041224 of gene with infection among sickle cell anemia Tunisian patients

Background and aimSickle cell anemia (SCA) is characterized by variable patterns of clinical expression. Polymorphisms linked to different genes have been associated with specific complications of the disease. Herein, we focused on the study of the association of 4 polymorphisms of Insulin like Growth Factor 1 receptor (IGF1R) gene with infections, which are the major cause of death in SCA.Material and methodsThis study involved 116 sickle cell patients among whom 58 SS have the same confirmed infectious phenotype. Allele-Specific PCR was performed for the study of rs1319868, whereas the PCR/sequencing method was carried out for rs1567811, rs2872060 and rs8041224.ResultsThe results showed that rs1319868 and rs1567811 were associated with a decreased risk of infection among SS patients (p=0.038, RR=0.54; p=0.044, RR=0.56, respectively). Interestingly, the combination of different genotypes showed the association of the genotype GT of rs1319868 and the genotype CC of rs8041224 with further decreased infection risk in SCA (p=0.028, RR=0.04).ConclusionThese significant associations of IGF1R SNPs with infection suggest that this gene could play an important role in the immune function in SCA