Slaheddine Fattoum

Molecular Basis of β‐Thalassemia in the Population of Tunisia

The present study attempts to delineate the spectrum of β‐thalassemia (thal) mutations in Tunisia by studying a large population from different parts of the country. A total of 285 unrelated subjects, 190 of whom had β‐thal major, 72 with Hb S/β‐thal, one with Hb C/β‐thal, one with Hb O‐Arab/β‐thal and 21 β‐thal carriers, were studied. The molecular defects were detected in 97.7% of the β‐thalassemic chromosomes (n = 475). Nineteen different β‐thalassemic alleles were identified. Two mutations, namely codon 39 (C→T) and IVS‐I‐110 (G→A) accounted for 70.0% of the studied chromosomes, followed by IVS‐I‐1 (G→A) (4.5%). Five other mutations, frameshift codon (FSC) 44 (–C), codon 30 (G→C), IVS‐I‐2 (T→G), IVS‐II‐745 (C→G), and FSC 6 (–A), are not uncommon in this population, while the remaining 11 mutations, IVS‐I‐5 (G→A), − 30 (T→A), codons 25/26 (+ T), IVS‐I‐6 (T→C), FSC 5 (–CT), IVS‐II‐848 (C→A), FSC 8 (–AA), –87 (C→G), IVS‐I‐5 (G→C), IVS‐II‐1 (G→A) and IVS‐II‐849 (A→C) are quite rare; four of these have not been previously reported in the Tunisian population. Potential origin and spread of these mutations to Tunisia are also discussed.

Distribution of CFTR Mutations in Cystic Fibrosis Patients of Tunisian Origin: Identification of Two Novel Mutations

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). More than 500 molecular defects have been reported to date. The distribution of these mutations is both heterogeneous and population related. In Mediterranean populations, 20–30% of CF alleles remain unidentified. We have studied a sample of 39 CF patients of Tunisian origin and have used a GC clamp DGGE assay to scan the CFTR gene. Two novel mutations have been found, but we have been unsuccessful in finding any mutation in 40% of these alleles. These results suggest that, in this Mediterranean population, additional mutations may lie elsewhere in the promoter region or in introns not yet analyzed.

Cystic Fibrosis Transmembrane Conductance Regulator Mutation Spectrum in Patients with Cystic Fibrosis in Tunisia

AIM To determine the frequency and types of mutations causing cystic fibrosis (CF) in Tunisia. METHODS We analyzed the complete coding region and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 68 unrelated patients suffering from the classical form of the disease. RESULTS Twelve different CFTR mutations accounted for 90% (123/136) of CF alleles, including F508del (47.06%), E1104X (16.18%), N1303K (6.62%), 711 + 1T > G (5.88%), W1282X (4.41%), G542X (3.67%), R1158X (1.47%), 4016insT (0.74%), and R785X (0.74%). Three novel mutations were detected in this study: I1203V (1.47%), 1811 + 5A > G (0.74%), and 4268 + 2T > G (1.47%). Fifty patients (74%) were homozygous, among which 28 (41.17%) for F508del and 10 (14.7%) for E1104X. CONCLUSIONS Ninety-seven percent of patients were found with at least one CFTR mutation. This study contributes to a better knowledge on CF-causing mutations in different regions in Tunisia and demonstrates that a complete scanning of CFTR sequences is necessary to implement efficient programs for CF genetic screening and counseling in this part of North Africa.

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.

The erythrocyte effects of haemoglobin OARAB

To test the hypothesis that HbOARAB induces an increase in red cell mean corpuscular haemoglobin concentration (MCHC), we studied members of four Tunisian families who were either homo‐ or heterozygous for HbOARAB or were double heterozygotes for HbS and HbOARAB. The α‐gene status was also tested. The findings included: (1) Distinctive variation in red cell density (MCHC) as determined by separation of red cells on isopycnic gradients: (a) All red cells from patients homozygous for HbOARAB were denser than normal red cells, as is observed for homozygous HbC patients. (b) In patients heterozygous for HbOARAB, red cell density was strongly influenced by the presence of α‐thalassaemia. The coexistence of −α/αα resulted in an average red cell density slightly greater than normal (AA) red cells. Patients heterozygous for HbOARAB with a normal complement of four α genes had denser red cells similar to sickle cell disease with some cells of normal density but with most cells very dense. (c) Finally, the double heterozygotes for HbS and HbOARAB had significant haemolytic anaemia and red cells denser than normal with some as dense as the densest cells found in sickle cell anaemia. (2) Reticulocytes in patients homozygous for HbOARAB were found in the densest density fraction of whole blood. (3) Cation transport in patients homozygous for HbOARAB was abnormal, with K:Cl cotransport activity similar to that of HbS‐Oman and only somewhat lower than in sickle cell anaemia red cells. The activity of the Gardos channel was indistinguishable from that found in HbS, HbC and HbS‐Oman cells.

Detection of Two Rare β-Thalassemia Alleles Found in the Tunisian Population: Codon 47 (+A) and Codons 106/107 (+G)

We here present the first report of the detection of two rare β0-thalassemia (thal) mutations in the Tunisian population: codon 47 (+A) and codons 106/107 (+G). To the best of our knowledge this is the second report of the codon 47 (+A) mutation, the first being identified in a Surinamese subject. The codons 106/107 (+G) mutation was first described in American Blacks, subsequently in Egyptians and Palestinians, and now in Tunisians. These mutations were detected by denaturing gradient gel electrophoresis (DGGE) screening followed by automated nucleotide sequencing. The former was found in two related β-thal major patients in the homozygous state, while the latter was identified in a homozygous state in a transfusion-dependent β-thal subject and in a sickle cell β-thal patient. Both mutations are in linkage disequilibrium with haplotype V and sequence framework 2. Given the known wide spectrum of β-thal alleles in the Tunisian population, the present report further confirms such heterogeneity. The knowledge of an updated spectrum of β-thal alleles in Tunisia must allow the implementation of a more efficient screening strategy for genetic counseling and prenatal diagnosis.

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.

Haplotypes Linked to Three Rare β-Thalassemia Mutations, Originally Reported in Tunisia

The polymorphism of the β-globin gene haplotypes and frameworks are useful in the determination of the unicentric and multicentric origin of a mutational event. In order to improve our knowledge of the chromosomal background of the β-globin gene in three β-thalassemia (thal) mutations originally reported in Tunisia, namely codons 25/26 (+T), codon 30 (G→C) and IVS-I-2 (T→G), we have investigated 13 unrelated individuals. There were five non transfusion-dependent patients homozygous for the IVS-I-2 (T→G) mutation, five others were homozygous for the codon 30 (G→C) mutation, one was a homozygote for the codons 25/26 (+T) insertion mutation and one patient was a compound heterozygote for the codon 39 (C→T) and codon 25/26 (+T) mutations; the last patient had a βS/codon 25/26 (+T) compound heterozygous genotype. Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based methods. The framework polymorphism was established by direct sequencing. β-Globin gene analyses demonstrated that all IVS-I-2 (T→G) cases were associated with haplotype IX; the codon 30 (G→C) mutation was supported by haplotype I, while the codons 25/26 (+T) mutation was linked to haplotypes I and IX.

A Novel α‐Thalassemia Nonsense Mutation in Codon 23 of the α2‐Globin Gene (GAG→TAG) in a Tunisian Family

Herein we describe a novel α‐thalassemia (thal) point mutation in the α2‐globin gene, found in a 3‐year‐old Tunisian girl who had Hb Barts (γ4) at birth, later on presenting with moderate anemia, microcytosis and hypochromia. She had a normal Hb A2 level and no abnormal hemoglobin (Hb) fraction. After excluding most of the common Mediterranean mutations, the α2‐globin gene was sequenced and found to have a point mutation in the heterozygous state that creates a premature stop signal for translation (GAG→TAG or Glu→Term) at codon 23. The same mutation was also found in the mother in the heterozygous state, while the father had a normal sequence. The presence of the mutation was also confirmed by nucleotide sequencing of the opposite strand. Since the mutation creates a restriction site for the BfaI enzyme, a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP)‐based assay was established for screening purposes.

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Background: There are few data on the molecular basis of Cystic Fibrosis (CF) in North Africa, probably due to under-diagnosis. Aim: This is the first study of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in the Libyan population. Subjects and methods: This study analysed the complete coding region and flanking intronic sequences of the CFTR gene in 10 unrelated Libyan CF patients. Results: This study identified four mutations (F508del, c.1670delC, N1303K and E1104X), with a high frequency of the latter. Conclusion: Identification of CF mutations facilitates molecular investigation of cystic fibrosis in the Libyan population and helps to provide effective genetic counselling among CF families.