Faida Ouali

First Description in Tunisia of a Point Mutation at Codon 119 (CCT→TCT) in the α1-Globin Gene: Hb Groene Hart in Association with the − α3.7 Deletion

Herein we describe the case of a Tunisian girl who presented with 3% Hb Barts (γ4) at birth. At the age of 3 years, she showed microcytosis and hypochromia in the absence of iron deficiency. The first step of molecular analysis was to test for the common Mediterranean mutations and the classical − α3.7 deletion was found in the heterozygous state. Since this finding could not explain the level of Hb Barts at birth, or the hypochromia and microcytosis, all the α-globin genes were sequenced. This revealed a rare point mutation at codon 119 (CCT→TCT) in the α1-globin gene, identified for the first time in Tunisia, and which has previously been described as an unstable hemoglobin (Hb) variant named Hb Groene Hart [α119(H2)Pro→Ser (α1)]. Here the − α3.7/αα119(CCT→TCT) genotype is responsible for the α-thalassemia (thal) trait phenotype.

Red cell indices: differentiation between β-thalassemia trait and iron deficiency anemia and application to sickle cell disease and sickle cell thalassemia.

BACKGROUND In Tunisia, thalassemia and sickle cell disease (SS) represent the most prevalent monogenic hemoglobin disorders with 2.21% and 1.89% of carriers, respectively. This study aims to evaluate the diagnosis reliability of 12 red blood cell (RBC) indices in differentiation of β-thalassemia trait (β-TT) from iron deficiency anemia (IDA) and between homozygous SS and sickle cell thalassemia (ST). METHODS The study covered 384 patients divided into three groups. The first one is composed of 145 control group, the second consists of 57 β-TT and 52 IDA subjects and the last one with 88 SS and 42 ST patients. We calculated sensitivity, specificity, positive-predictive values, negative-predictive values, percentage of correctly identified patients and Youdens Index (YI) for each indice. We also established new cut-off values by receiver operating characteristic curves for each indice. An evaluation study was performed on another population composed of 106 β-TT, 125 IDA, 31 SS, and 17 ST patients. RESULTS Srivastava Index (SI) shows the highest reliability in discriminating β-TT from IDA at 5.17 as a cut-off and also SS from ST with 7.7 as another threshold. Mentzer Index (MI) and RBC appear also useful in both groups with new cut-offs slightly different from those described in literature for β-TT and IDA. CONCLUSIONS The effectiveness and the simplicity of calculation of these indices make them acceptable and easy to use. They can be relied on for differential diagnosis and even for diagnosis of β-TT with atypical HbA₂ levels.

Prenatal diagnosis of cystic fibrosis: 10-years experience.

PURPOSE We present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. PATIENTS AND METHODS Based on family history, 40 Tunisian couples were selected for prenatal diagnosis. Fetal DNA was isolated from amniotic fluid collected by transabdominal amniocentesis or from chronic villi by transcervical chorionic villus sampling. The genetic analysis for cystic fibrosis mutations was performed by denaturant gradient gel electrophoresis and denaturing high-pressure liquid phase chromatography. We performed microsatellites analysis by capillary electrophoresis in order to verify the absence of maternal cell contamination. RESULTS Thirteen fetuses were affected, 21 were heterozygous carriers and 15 were healthy with two normal alleles of CFTR gene. Ten couples opted for therapeutic abortion. The microsatellites genotyping showed the absence of contamination of the fetal DNA by maternal DNA in 93.75%. CONCLUSION Our diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis.

δ0-Thalassemia in cis of βKnossos globin gene: first homozygous description in thalassemia intermedia Libyans and first combination with codon 39 (C→T) in thalassemia intermedia Tunisian patients.

Abstract Background: β-Thalassemia is the most common disease among hemoglobinopathies in North African and Arab populations. In the present study we report the first description of the β-Knossos codon27 (G→T) (βKnossos) allele in cis with the δ059 (-A) mutation in thalassemia intermedia patients in Tunisia and Libya. Methods: This identification was carried out by sequencing analysis of the whole coding regions of the δ- and β-globin genes. Results: We noted that heterozygous inheritance of the βKnossos mutation results in a mild β-thalassemia phenotype with a low level of HbA2 while homozygous leads to intermediate β-thalassemia with an atypical high performance liquid chromatogram showing a complete absence of HbA2 and HbF. Compound heterozygosity of the βKnossos with β0 codon39 (C→T) is identified in a Tunisian proband for the first time and gives rise to a mild phenotype. In both families, the δ0 codon59 (-A) and the βKnossos alleles were found to be associated with a single Mediterranean β-haplotype I similar to that observed in previous reports from Algeria, Egypt, Cyprus, and Turkey. Conclusions: The chromosome supporting the βKnossos and the δ0 codon59 (-A) alleles seems to be of a single Mediterranean origin. Premarital screening studies in families in which only one of the parents has typical aspects of β-thalassemia trait and the other has a normal HbA2 level associated with abnormal red cell indices becomes a necessity to avoid missing thalassemia carriers.

Determination of glucose-6-phosphate dehydrogenase cut-off values in a Tunisian population.

Abstract Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest enzymopathy worldwide. The incidence depends essentially on the methods used for the assessment. In this respect, we attempted in this study to set cut-off values of G6PD activity to discriminate among normal, heterozygous, and deficient individuals using the World Health Organization (WHO) classification and the receiver operating characteristics (ROC) curve analysis. Methods: Blood samples from 250 female and 302 male subjects were enrolled in this study. The G6PD activity was determined using a quantitative assay. The common G6PD mutations in Tunisia were determined using the amplification refractory mutation system (ARMS-PCR) method. The ROC curve was used to choice the best cut-off. Results: Normal G6PD values were 7.69±2.37, 7.86±2.39, and 7.51±2.35 U/g Hb for the entire, male, and female groups, respectively. Cut-off values for the total, male, and female were determined using the WHO classification and ROC curves analysis. In the male population, both cut-offs established using ROC curve analysis (4.00 U/g Hb) and the 60% level (3.82 U/g Hb), respectively are sensitive and specific resulting in a good efficiency of discrimination between deficient and normal males. For the female group the ROC cut-off (5.84 U/g Hb) seems better than the 60% level cut-off (3.88 U/g Hb) to discriminate between normal and heterozygote or homozygote women with higher Youden Index. Conclusions: The establishment of the normal values for a population is important for a better evaluation of the assay result. The ROC curve analysis is an alternative method to determine the status of patients since it correlates DNA analysis and G6PD activity.

Prenatal diagnosis of hemoglobinopathies in Tunisia: an 18 years of experience

Hemoglobinopathies are the most common genetic disease in Tunisia with a total carrier prevalence of 4.48%.

Thrombophilic Mutations Among Patients with Sickle Cell Disease

BACKGROUND Factor V-Leiden (FVL), Prothrombin (PRT) G20210A, and Methylene Tetrahydro Folate Reductase (MTHFR) C677T and A1298C mutations are major inherited risk factors of thrombotic complications. Our aim in this study was to investigate the prevalence of these mutations among Tunisian sickle cell patients. METHODS Study subjects comprised 64 patients and 100 healthy controls. FVL, PRT G20210A, and MTHFR genotypes were determined using a reverse dot blot based method. RESULTS In the patient population studied, the prevalence of FV Leiden was not statistically different from controls while a significant prevalence of heterozygous PRT G20210A mutation among patients (10.93%) was found. An increased frequency of the MTHFR 677 C>T genotype was seen among patients as well as controls. The results showed no significant association between the MTHFR A1298C mutation and sickle cell disease (SCD). However, the prevalence of carrier among studied patients was 15.62% compared to 7% among healthy subjects. CONCLUSIONS In conclusion, our data suggest a significant association between PRT G20210A and MTHFR C677T and sickle cell disease among Tunisian patients.

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

Abstract 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 β-thalassemia (β-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of β-thal major (β-TM) and moderate type of β-thal intermedia (β-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% β-TI patients, which was not the case in the β-TM patients or in the control group. Thus, the T allele is consequently associated with the β-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 β-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.

Prenatal Diagnosis of Hemoglobinopathies: A Case Study on Tunisia

Hemoglobinopathies are the most common genetic disease in Tunisia with a total carrier prevalence of 4.48%, reaching 12.5% at certain affected regions. The β-thalassemia trait frequency is 2.21% and the sickle cell trait is 1.89%. A prenatal diagnosis (PND) unit has been progressively installed since 1986 at the biochemistry and molecular biology department of children’s hospital of Tunis. The present study tries to explore the prenatal diagnosis of the Hemoglobinopathies in Tunisia for the period of 1994-2012 and tries to share its experiences and the progress achieved in overcoming this challenge. 340 out of 461 fetuses examined for this study were at risk and couples that have agreed for pre-diagnosis have benefited a lot in averting the major health risks like beta-thalassemia (41%), sickle cell anemia (40.3%), S/beta-thal (14.7%) and the remaining fetuses were at risk for a compound heterozygote hemoglobinopathies (S/O, O/beta-thal, S/C). 25.8% of the couple covered in this study have voluntarily asked for PND several times, as they were worried about giving birth to a child with Hemoglobinopathy. Fetal DNA was collected from chorionic villis biopsy of 53 cases, amniotic fluid samples are considered in 397 cases and CVS was followed in 7 cases for lack of results. Out of 461 tested foetuses, 26.2% of them were affected, 50.5% were carriers of the disease and 19.3% were completely healthy. PND fail to detect the problem in 3.9% of the cases. Except 13% of the affected fetuses, the entire number of cases detected with defected fetuses had been aborted. Although abortion is legal in Tunisia, 13% of pregnant women with affected fetus have refuted to abortion due to religious reasons. The total number of fetus aborted remained 1.53%. Although PND was successful in our lab, it was insufficient to cover the entire Tunisian territory. The researchers hence recommend to the health authorities to establish widespread PND services in places that are worst affected with these cases in Tunisia to prevent hemoglobinopathies