Homozygous Mutation on the ß-Globin Polyadenylation Signal in a Tunisian Patient with ß-Thalassemia Intermedia and Coinheritance of Gilbert's Syndrome.

We report here the clinical, hematological and molecular data in a 50-year-old patient with ß-thalassemia intermedia (ß-TI) caused by a homozygous ß+ mutation on the ß-globin gene polyadenylation (polyA) signal (AATAAA>AAAAAA). ß Haplotype analysis was accomplished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype and framework analysis showed that this mutation is associated with the [- - - - + + +] ß haplotype and framework 1 (CCGCT) (FW1). This mutation was previously reported in the heterozygous state in association with the codon 9 (+TA) mutation in a ß-TI patient originating from Tunisia. To the best of our knowledge, this is the first report describing this mutation in the homozygous state. The case reported here, coinherited Gilbert's syndrome, which is characterized by hyperbilirubinemia. This conclusion was reached by the investigation of the promoter region [A(TA)nTAA] motif of the UGT1A1 gene, showing the (TA)6/(TA)7 genotype.

First description of the rs45496295 polymorphism of the C/EBPE gene in ß-thalassemia intermedia patients.

The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of ß-thalassemia (ß-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of ß-thal major (ß-TM) and moderate type of ß-thal intermedia (ß-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% ß-TI patients, which was not the case in the ß-TM patients or in the control group. Thus, the T allele is consequently associated with the ß-TI group (p = 10-3). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10-3) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of ß-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.

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

AIMS: 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' and '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 × 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.

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

AIMS: 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' and '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 × 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.

Gilbert syndrome acts as a risk factor of developing gallstone among ß hemoglobinopathy Tunisian patients.

BACKGROUND: As a result of chronic hemolysis, hyperbilirubinemia is often observed, leading to the formation of pigment cholelithiasis which could be busted by the presence of uridine diphosphoglucuronosyltransferase 1A1 defects. AIM: Herein, we investigated the effect of glibert mutation on the occurrence of pigment cholelithiasis in Tunisian patients with beta (ß) hemoglobinopathy including sickle cell anemia and ß thalassemia (minor). SUBJECTS AND METHODS: Our study included 151 subjects divided in 75 SCA patients and 76 ß thalassemia patients. Both groups of patients were divided into two sub-groups according to the presence or absence of cholelithiasis. The relationship between A(TA)nTAA variation of UGT1A1 gene, the serum bilirubin level and the occurrence of cholilithiasis was investigated. RESULTS: Our results show a significant association between genotypes carrying variant (TA)7 and hyperbilirubinemia (p<0.05). Furthermore, we demonstrated a significant association between (TA)6/(TA)7 and (TA)7/(TA)7 genotypes with cholelithiasis among sickle cell anemia and thalassemia patients (p<0.05). CONCLUSION: Altogether, our data provide evidence that genotypes (TA)6/(TA)7 and (TA)7/(TA)7 and (TA)7 variant present a risk factor of developing gallstone among ß hemoglobinopathy Tunisian patients.

Monocentric study of Willebrand's disease in Tunisia: assets and difficulties.

BACKGROUND: Von Willebrand's disease (VWD) is the most commonly inherited bleeding disorder. It is characterized by clinical, biological and molecular heterogeneity. In certain types of the disease, diagnosis can be difficult. AIM: We report the clinico-biological characteristics of VWD's patients and analyze diagnosis difficulties. METHODS: 33 cases were diagnosed in the laboratoryfrom February to May, 2011. Screening hemostasis included the measuring of FVIII: C, VWF: Ag and VWF: RCo. Blood cell count and blood group were performed in all cases. RESULTS: Mean age at diagnosis is 13 years [10 months -43 years]. The sex ratio M/F is 0.5. The patients are classified type 3 VWD in 52% of the cases, type 2 VWD in 30 % of the cases and type 1 VWD in 18 % of the cases. The diagnosis of type 2B VWD suspected in combination of the ratio VWF:RCo / VWF: Ag <0,7 and thrombocytopenia in one case. Required tests for positive diagnosis and distinction between the primary categories of VWD are available. Specialized tests will allow a best characterization variants type 2 VWD for a better therapeutic approach.

Association between rs267196 and rs267201 of BMP6 gene and osteonecrosis among sickle cell aneamia patients.

AIMS: The skeletal manifestations of sickle cell disease are the result of changes in bone and bone marrow caused by chronic tissue hypoxia that is exacerbated by episodic occlusion of the microcirculation by the abnormal sickle cells. Furthermore, the occurrence of osteonecrosis is under the control of some modifier gene. BMP6 (Bone morphogenetic protein) has been reported as associated with osteonecrosis in sickle cell anemia (SCA). Herein, we intend to study the impact of rs267196, rs267201, rs408505 and rs449853 of BMP6 gene in the occurrence of osteonecrosis among sickle cell patients in Tunisia. METHODS: Our study involved 100 SCA patients among whom 19 have osteonecrosis of the head of the femur. The latter polymorphisms of BMP6 gene were analyzed for all subjects by PCR/sequencing. To test for trait association with the candidate SNPs, genotype and allele frequencies between cases (osteonecrosis group) and controls (non-osteonecrosis group) were compared using Pearson's chi_square test with a significance threshold of P<0.05 (compare 2, version 1.02). RESULTS: Our findings showed that the patients carried genotype TA of rs 267196 and genotype AG of rs267201 present a high risk factor for developing osteonecrosis RR=1.317 and RR=1.3 respectively. The results showed a significant association between the alleles A of rs 267196 and G of rs267201 and osteonecrosis P=0.0023; RR=2.42 and P=0.041; RR=2.24 respectively. Interestingly, SCA patients with the combined genotype TA/AG were found to be at higher risk of developing osteonecrosis (P=0.009). As for rs408505 and rs449853 of BMP6 gene no significant association was found among SCA patients.

[Immunophenotyping of B chronic lymphoproliferative syndromes (CLL excluded): confrontation with the histology]. / Immunophénotypage des syndromes lymphoprolifératifs chroniques B non LLC : confrontation avec l'histologie.

Immunophenotyping is a major tool for the diagnosis of the chronic lymphoïd leukaemia (CLL). Its interest remains limited in the classification of the other B chronic lymphoproliférative syndromes (B-CLPS). We evaluate the place of the flow cytometry (CMF) in the diagnosis and classification of the non CLL B-CLPS. The cases with Matutes score of 4 or more are excluded. A confrontation of the results to the histology is made. 28 cases of non CLL B-CLPS are diagnosed. CMF shows a κ monoclonal population in 15 cases and λ in 13 cases. A co-expression CD19+CD5 + is found in 11 cases concording with an atypic CLL or a mantel cell lymphoma in 6 cases with Matutes score of 3. In 5 cases, we concluded to non CLL B-CLPS (Matutes<3). The histology retained the diagnosis of a mantel cell lymphoma (4 cases), a SLVL (1 case) and an atypical LLC (1 case). CD5 is negative in 17 cases. In 5 cases, the diagnosis of hairy cell leukemia (HCL) is retained (CD 11c+ CD103+) and confirmed by the histology. The diagnosis of a marginal zone lymphoma is retained in 2 cases, a SLVL in 2 cases, a follicular lymphoma in 3 cases and prolymphocytes leukaemia in 1 case. Nine cases of non CLL B-CLPS were difficult to classify by histology. CMF is insufficient for the classification of most of the non CLL B-CLPS. Only the phenotype of the HCL is characteristic. The confrontation of the histology results remains essential.

Two new class III G6PD variants [G6PD Tunis (c.920A>C: p.307Gln>Pro) and G6PD Nefza (c.968T>C: p.323 Leu>Pro)] and overview of the spectrum of mutations in Tunisia.

We screened 423 patients referred to our laboratory after hemolysis triggered by fava beans ingestion, neonatal jaundice or drug hemolysis. Others were asymptomatic but belonged to a family with a history of G6PD deficiency. The determination of enzymatic activity using spectrophotometric method, revealed 293 deficient (143 males and 150 females). The molecular analysis was performed by a combination of PCR-RFLP and DNA sequencing to characterize the mutations causing G6PD deficiency. 14 different genotypes have been identified : G6PD A(-) (376A>G;202G>A) (46.07%) and G6PD Med (33.10%) were the most common variants followed by G6PD Santamaria (5.80%), G6PD Kaiping (3.75%), the association [c.1311T and IVS11 93c] (3.75%), G6PD Chatham (2.04%), G6PD Aures (1.70%), G6PD A(-) Betica (0.68%), the association [ 376G;c.1311T;IVS11 93c] (0.68%), G6PD Malaga, G6PD Canton and G6PD Abeno respectively (0.34%). Two novel missense mutations were identified (c.920A>C: p.307Gln>Pro and c.968T>C: p.323 Leu>Pro). We designated these two class III variants as G6PD Tunis and G6PD Nefza. A mechanism which could account for the defective activity is discussed.

[Immunophenotyping in adult acute myeloid leukemia: which prognostic value?]. / Immunophénotypage des leucémies aiguës myeloïdes de l'adulte : quelle valeur pronostique ?

BACKGROUND: Immunophenotyping is an essential step in the diagnosis of acute myeloid leukemia. Its prognostic value remains controversial with contradictory results. AIM: To assess prognostic impact of the immunophenotyping in AML. METHODS: Our study is retrospective (October, 2005 - July, 2007) concerning 56 cases of AML (AML3 excluded) of the adult from 18 to 55 years old diagnosed and treated in Tunis Aziza Othmana Hospital. The immunophenotyping was performed by flow cytometry (Beckman Coulter EPICS XL MCL®). We studied clinical and biological characteristic, immunophenotypic expressions, and parameters of the response to the treatment: complete remission (CR), overall survival (OS), relapse free survival (RFS) and relapse in a delay of 1 year and 2 years. SPSS software was used to perform the statistical analysis. RESULTS: The median age of the patients is of 37,7±11.8 years. Sex ratio (M/F) is 1.33. Among individual antigenic expressions, only CD7 is associated to lower CR rates (p=0.044). We did not find any statistically significant association between immunophenotypic expressions and OS nor with relapse or RFS. CONCLUSION: The impact of immunophenotyping in AML remains controversial because of contradictory results. The research of molecular changes would be an interesting alternative in our context.

Treatment of Acute Promyelocytic Leukemia with AIDA Based Regimen. Update of a Tunisian Single Center Study.

In Tunisia, the ATRA era began in 1998 with the use, consecutively, of two regimens combining ATRA and an anthracycline with cytarabine (APL93), and without cytarabine (LPA99). From 2004, 51 patients with confirmed APL either by t(15;17) or PML/RARA were treated according to the PETHEMA LPA 99 trial. Forty three patients achieved CR (86%). The remaining seven patients had early death (one died before treatment onset): four caused by differentiation syndrome (DS) and three died from central nervous system hemorrhage. Multivariate analysis revealed that female gender (P=0.045), baseline WBC> 10 G/L (P=0.041) and serum creatinine > 1.4mg/dl (P=0.021) were predictive of mortality during induction. DS was observed in 16 patients (32%) after a median onset time of 15 days from treatment onset (range, 2-29). Body mass index ≥ 30 (P=0.01) remained independent predictor of DS. Occurrence of hypertensive peaks significantly predicted occurrence of DS (P=0.011) and was significantly associated with high BMI (p=0.003). With a median follow-up of 50 months, 5 year cumulative incidence of relapse, event free and overall survival were 4.7%, 74% and 78%, respectively.

[Iron overload in sickle cell anemia : a study of 94 patients]. / Évaluation de la surcharge en fer au cours de la drépanocytose : à propos de 94 cas.

BACKGROUND: Sickle cell disease is an autosomal, recessive hemoglobinopathy characterized by hemolytic anemia. Red blood cell transfusions are uncommon therapeutic mainstay in sickle cell disease and repeated transfusions can result in iron overload. The predicted risks of iron overload and organ failure increase with both the duration of disease requiring transfusion therapy and the number of transfusions. AIM: To assess the state of iron overload in patients with sickle ce anemia according to their number of transfusions. METHODS: The medical records of 94 patients with sickle cell anemia (46 had homozygous sickle cell disease, 41 had sickle-ß thalassemia, 7 had compound heterozygous hemoglobin: 4 SC and 3 SOArab) were retrospectively reviewed for the following: clinical exam, serum ferritin level, liver function tests, abdominal ultrasound exam and heart Doppler. RESULTS: 61% of our patients are from the Northern- west of the country. The average age is 18.29 years (2 to 62 years) and the sexratio is 0.62. In addition to parental consanguinity which is found in 28.72% of the cases. The average level of ferritin is 660.35 ng/ml. 41.5% of the patients have a high status of ferritin witch ranged from 521.4 to 3360 ng/ml. There is not a significant difference of ferritin level according to age, sex and a phenotype of sickle cell anemia. However, it is higher among the transfused patients with a same phenotype (p<0.05). We found a correlation between serum ferritin levels and the number of transfusions (r =+0.74). Splenectomy has a preventive role because it allowed stopping the transfusion in 65% of the cases. The evaluation of organ dysfunction has found a hepatomegaly in 29% of the cases, half of witch were have a high status of serumferritin (> 1000 ng/ml). Left ventricular hypertrophy associated to valvulopathy was classified in 10 % of the cases. CONCLUSION: Iron overload in sickle cell anemia, though relying on transfusion, remains moderate. The repetitive assessment of serum ferritin level is considered as the best test though it does not evaluate an organic dysfunction. To evaluate them better, other tests are requiring: magnetic resonance imaging and Tc-Squid biosusceptometers.

[Thalassemia intermedia: 36 cases]. / La bêta thalassémie intermédiaire: a propos de 36 cas.

BACKGROUND: Thalassemia intermedia empasses a mild clinical and biological spectrum. The aim is to report the clinical and biological features and treatment of this disease. METHODS: It is a retrospective study about 36 thalassemia intermedia patients (17 males, 19 females). Epidemiological, haematological aspects and treatment were reported RESULTS: The diagnosis was carried out at a relatively old age 15 years (1-72).The thalassemia intermedia was characterized by mild facial deformities, splenomegaly and moderate anemia ( Hb = 9.1 g/dl). The mean serum ferritin was 518 ng/ml (25-1800). Three phenotypes are caracterised: heterozygosis beta thalassemia, beta degrees thalassemia and beta + thalassemia. Clinical complications were hypersplenism, extra medullary hematopoiesis, leg ulcers, thrombosis and pulmonary hypertension. Treatment was based on occasionally transfusion and splenectomy on event of hypersplenious (47%). Evolution of this disease was generally good with a long lifespan at 31 years (6-83). CONCLUSION: Thalassemia intermedia is well tolerated. Transfusions and splenectomy were indicated in case of hypersplenious.

[Splenectomy in hereditary hemolytic anemia: 82 Tunisian cases]. / La splenectomie dans les anemies hemolytiques constitutionnelles: a propos de 82 cas tunisiens.

BACKGROUND: Splenectomy is frequently advised in hereditary hemolytic anemia. Severe complications could occur after splenectomy. AIM: To provide the indication and benefit of splenectomy METHODS: clinical and biological patterns were performed in a retrospective study of 82 patients: 17 homozygous beta thalassemia, 17 thalassemia intermedia, 33 heterozygote Hb/S beta thalassemia and 15 hereditary spherocytosis. RESULTS: Splenectomy was performed for: Hypertransfusion in homozygous thalassemia, hereditary spherocytosis; hypersplenism in Thalassemia intermedia and splenic sequestration in heterozygote HbS/beta thalassemia. The benefit of splenectomy was proved in hereditary spherocytosis (100%), heterozygote HbS/beta thalassemia (90%) and thalassemia intermedia (75%); nevertheless in homozygous beta thalassemia. Post splenectomical complication are often thrombocytosis, thrombosis and infections. CONCLUSION: Splenectomy should be performed in hereditary hemolytic anemia to reduce and avoid transfusion.

[Soluble tranferrin receptor in the biological diagnosis of iron deficiency. Report of 24 cases]. / Apport du récepteur soluble de la transferrine dans le diagnostic biologique de la carence en fer chez l'homme. A propos de 24 cas.

BACKGROUND: In inflammatory diseases, classical parameters of iron status (serum iron, serum ferritin, total iron-binding capacity of transferrin and transferrin saturation) are not very reliable. AIM: The purpose of this study is to investigate soluble transferrin receptor, its index and classical parameters of iron status [serum iron, serum ferritin, total iron-binding capacity of transferrin and transferrin saturation] during iron-deficiency anemia and combined iron deficiency and inflammatory anemia. METHODS: Our study concerned 24 patients: 18 patients with iron-deficiency anemia and 6 patients with combined iron-deficiency and inflammatory anemia. 55 healthy subjects were included as controls. Both groups underwent classical parameters of iron status [serum iron, serum ferritin, total iron-binding capacity of transferrin and transferrin saturation] and measurement of soluble transferrin receptor with its index. RESULTS: In iron-deficiency anemia, total iron-binding capacity of transferrin, soluble transferrin receptor and its index were enhanced, whereas serum iron, ferritinemia and transferrin saturation were low compared to controls. Compared to patients with iron-deficiency anemia, those with combined iron-deficiency and inflammatory anemia showed higher levels of serum iron and ferritinemia. In contrast, soluble transferrin receptor and its index did not vary significantly between both groups. CONCLUSION: Our findings show the interest of soluble transferrin receptor and its index in the detection of iron deficiency during anemia of inflammatory states.

[Cytology and immunophenotype features of hairy cell leukemia: 6 cases]. / Caracteristiques cytologiques et immunophenotypiques des leucemies a tricholeucocytes: a propos de 6 cas.

BACKGROUND: Hairy cell leukemia is a rare lymphoproliferative disorder. AIM: with cytological and immunophenotypic features. METHODS: We report 6 cases of hairy cell leukemia diagnosed in the Biological Department of Hematology at the Aziza Othmana Hospital of Tunis. RESULTS: Hairy cells was observed in blood smears of 5 cases. Flow cytometry analysis shown monoclonal a monoclonal population B while gatinting on the expression of the CD19 and SSC signal. The positivity of the CD103 is noted in 5 cases and the CD11c signal is intense in all the cases. CONCLUSION: Immunophenotype is of great interest in the diagnosis of hairy cell leukemia.

[Hemoglobin C disease: report of 16 Tunisian cases]. / L'hemoglobinose C: a propos de 16 cas Tunisiens.

AIM: was to provide the clinical and biological patterns hemoglobine disease in Tunisia. METHODS: This retrospective study collected to 16 cases of hemoglobin C disease : 6 homozygotic Hb C and 10 heterozygotic Hb C/beta-thalassemia. RESULTS: The clinical profile is characterized by mild hemolytic anemia (Hb = 11.7 g/dl) associated with splenomegaly and hypersplenism. Contrary to homozygous state, the Hb C/beta-thalassemia is associated with microcytosis and pseudopolycythemia. The diagnosis is based on target cells, specific intraerythrocytic Hb C crystals in blood smear and Hb C level at 100%. CONCLUSION: The Hb C disease must be considered as a benign hemoglobinopathy which is associated with a long survival without major complications.