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

UNLABELLED: Hemoglobinopathies are the most common genetic disease in Tunisia with a total carrier prevalence of 4.48%. OBJECTIVE: The aim of this study was to report an 18-year fully achieved experience of prenatal diagnosis (PND) of hemoglobinopathies (1994-2012) and to assess the impact of this prevention program. PATIENT AND METHODS: A total of 461 fetuses of 340 at-risk couples have been the subject of PND for beta-thalassemia major risk (41%), for sickle cell anemia risk (40.3%), for S/beta-thal risk (14.7%). The remainder fetuses were at risk for a compound heterozygote hemoglobinopathies (S/O, O/beta-thal, S/C….). Fetal DNA was studied by PCR procedure including the reverse dot-blot technique and the amplification refractory mutation system and direct sequencing. RESULTS AND DISCUSSION: Only 13.8% of the fetal samplings were conducted by chorionic villus sampling. The molecular result for beta-thalassemia risk has shown 13 beta-thal mutations, with two common: codon 39 (C>T) and IVS1-110 (G>A). The last 3 years, STR study has permitted to reduce the problems of maternal cell contamination. Among the 461 tested fetuses, 121 were affected, and then the pregnancy was terminated except for 13 cases, because of religious considerations and this despite the abortion legality in Tunisia. The conducted PND is only about 30 PND per year corresponding essentially to the couples living in Tunis City and surrounding area. PND number has increased from 1994 to 2009. This evolution has brutally decreased after the Tunisian revolution (2010). CONCLUSION: Although the good running of the PND, it covers only the Tunis city with low impact because it prevent apparition of only a mean of 7.3% of new cases. The reduced number of PND is not a technical inconvenience but rather a lack of a preventive program.

Impaired oxygen uptake kinetics in the first high-level athlete with Hb Hope: a case study.

Hemoglobin (Hb) Hope is a beta-globin chain variant with reduced oxygen (O2) affinity, known to induce anemia. This usually leads to limitations in O2uptake (VO2) and exercise tolerance. We studied the case of a high-level female athlete with Hb Hope. She had been selected for cross-country races from 13 yrs onward, then was a national junior champion in 400-m race, and finally failed to win any cross-country races as an adult. Hematological analysis revealed normal red blood cell indices and Hb level (12.3 g.dL⁻¹). Incremental exercise showed peak work rate (WR), VO(2max) and gas exchange threshold (GET) within normal ranges for healthy females. Constant WR testing at 90% of GET showed that kinetics of pulmonary VO2included the presence of a slow component. This was in disagreement with the data on VO2kinetics response to exercise intensities below GET. Phase 2 parameters, time constant (τ2, 31 s), time delay (TD2, 39 s), amplitude (A2, 780 ml.min⁻¹), and gain in VO2(ΔVO2 .ΔWR-1, 9.2 ml.min-1.W⁻¹) were within normal ranges. Phase 3 showed a slow component similar to that reported in severe exercise. The absence of anemia and the normality of phase 2 suggested normal O2delivery and oxidative metabolism in exercising muscles. In contrast, phase 3 suggested poor aerobic capacity and limited exercise tolerance. However, the lack of symptoms during testing also suggested that the slow component was due to the specific recruitment of fast-twitch fibers in this former champion athlete with Hb Hope in races requiring mainly anaerobic metabolism.

Association between clinical expression and molecular heterogeneity in ß-thalassemia Tunisian patients.

Beta-thalassemia is the most frequent hereditary blood disorder in Tunisia because of its geographic localization and history. This pathology is characterized by a complex multisystem process with genetic and biochemical interactions. The aim of this work was to establish phenotype/genotype association through studying the distribution and the relationship between ß-thalassemia and α-thalassemia mutations and three polymorphic markers: the C → T polymorphism at -158 of the Gγ gene, the RFLP haplotype and the repeated sequence (AT)xTy in the ß globin silencer, in two groups of ß-thalassemia major and ß-thalassemia intermedia (TI) patients. Statistical analysis has shown that moderate expression seen in TI patients was significantly associated to ß(+) -87 (C → G), -30 (T → A) and IVSI-6 (T → C) mutations, haplotypes VIII, IX and Nb and to XmnI polymorphism. The regression analysis of combined genotypes (mutation/XmnI/RFLP haplotype) revealed that they contribute to justify 17.1 % of clinical expression diversity (p < 0.05). Among the studied genotypes the XmnI polymorphism seems to be the most determinant modulating factor, followed by the ß-thalassemia mutation and RFLP haplotype. Our findings highlight the heterogeneity of molecular background of ß-thalassemia that would be responsible of clinical variability.

Glucose-6-phosphate dehydrogenase deficiency in Tunisia: molecular data and phenotype-genotype association.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. In this study, we aimed to perform a molecular investigation of G6PD deficiency in Tunisia and to associate clinical manifestations and the degree of deficiency with the genotype. A total of 161 Tunisian subjects of both sexes were screened by spectrophotometric assay for enzyme activity. Out of these, 54 unrelated subjects were selected for screening of the most frequent mutations in Tunisia by PCR/RFLP, followed by size-based separation of double-stranded fragments under non-denaturing conditions on a denaturing high performance liquid chromatography system. Of the 56 altered chromosomes examined, 75 % had the GdA(-) mutation, 14.28 % showed the GdB(-) mutation and no mutations were identified in 10.72 % of cases. Hemizygous males with GdA(-) mutation were mostly of class III, while those with GdB(-) mutation were mainly of class II. The principal clinical manifestation encountered was favism. Acute hemolytic crises induced by drugs or infections and neonatal jaundice were also noted. Less severe clinical features such as low back pain were present in heterozygous females and in one homozygous female. Asymptomatic individuals were in majority heterozygote females and strangely one hemizygous male. The spectrum of mutations seems to be homogeneous and similar to that of Mediterranean countries; nevertheless 10.72 % of cases remain with undetermined mutation thus suggesting a potential heterogeneity of the deficiency at the molecular level. On the other hand, we note a better association of the molecular defects with the severity of the deficiency than with clinical manifestations.

alpha-Thalassaemia in Tunisia: some epidemiological and molecular data.

Unlike the other haemoglobinopathies, few researches have been published concerning alpha-thalassaemia in Tunisia. The aim of the present work is to acquire further data concerning alpha-thalassaemia prevalence and molecular defects spectrum in Tunisia, by collecting and studying several kinds of samples carrying alpha-thalassaemia. The first survey conducted on 529 cord blood samples using cellulose acetate electrophoresis, have displayed the prevalence of 7.38% Hb Bart's carriers at birth. Molecular analyses were conducted by PCR and DNA sequencing on 20 families' cases from the above survey carrying the Hb Bart's at birth and on 10 Hb H diseased patients. The results showed six alpha-globin gene molecular defects and were responsible for alpha-thalassaemia: -alpha(3.7), - -(MedI), alpha(TSaudi), alpha(2)(cd23GAG->Stop), Hb Greone Hart: alpha(1)(119CCT->TCT) corresponding to 11 genotypes out of which two are responsible for Hb H disease (- -(Med)/-alpha(3.7)) and (alpha(TSaudi)alpha/alpha(TSaudi)alpha) and a newly described polymorphism: alpha+6C->G. The geographical repartition of alpha-thal carriers showed that the -alpha3.7 deletion is distributed all over the country, respectively the alpha(HphI) and alpha(TSaudi) seem to be more frequent in the central region of the northeast region. The haematological and clinical data showed a moderate phenotype with a late age of diagnosis for Hb H disease. This work had permitted, in addition to an overview on alpha-thalassaemia in the country, the optimization of protocols for alpha-thalassaemia detection in our lab, allowing further investigations concerning phenotype-genotype correlation in sickle cell disease or beta-thalassaemia.

[Difference albumin-transferrin interest in the iron deficiency detection in a cohort of 1288 schoolchildren in the district of Tunis]. / Intérêt de la dissociation albumine-transferrine (DAT) dans le diagnostic de la carence martiale dans une cohorte de 1,288 écoliers dans une région du grand Tunis.

Iron deficiency (ID) is the most common nutritional deficiency worldwide especially among young children, women in pregnancy and breastfeeding. This study was undertaken to assess the prevalence of ID in 1288 pupil ranging in age from 11 to 14 years. Haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), serum iron (Fe) serum transferrin (Trf), serum ferritin (Ft) and an inflammtory proteic profil (IPP) were measured. The IPP combines the analysis of protein variations: protein results are converted in percent of normal values referenced for the technique used. It has been suggested that on the protein profile, an increase in serum transferrin level compared to a normal serum albumin level (DAT: difference albumin-transferrin), appears early in the course of ID. Iron deficiency was defined by a low serum ferritin (< 15 ng/mL) and/or a pathologic DAT (> 28%). Approximately, 33.8% of children had Ft < 15 ng/mL and 12,8% had DAT > 28% while ferritin values were in the normal range. Diagnosis performance (sensitivity, specificity and diagnosis efficacy) of ferritin and DAT were compared to the performance of high serum transferrin receptor (sTfR) values in 2 populations presenting or not a biological inflammation. Only the diagnosis efficacy of DAT was constant in both situations. In conclusion, the serum ferritin concentration is the first indicator of body storage iron identifying ID, however normal or elevated values of ferritin may be difficult to interpret particulary in the presence of inflammation. sTfR and DAT values are thus reliable indicators of ID in such circumstances.

[Molecular epidemiology of cystic fibrosis in Tunisia]. / Epidémiologie moléculaire de la mucoviscidose en Tunisie.

Cystic fibrosis is the most frequent autosomal recessive genetic disease in North European population. This pathology seems to not be rare in Tunisia. On another hand, development of molecular biology techniques has largely contributed to implement the study of the different mutations in the CFTR gene where over 1,300 mutations were reported. Herein, we describe the strategy used to detect molecular defects responsible of cystic fibrosis on 390 children (383 families) in Tunisian population. Several techniques were performed for genotype diagnosis: DNA extraction was from peripheral blood. Polymerase chain reaction (PCR) and polyacylamide gel electrophoresis, and reverse dot blot procedures were used to detect known point mutations. Denaturant gradient gel electrophoresis (DGGE) were used in a next step searching for the unknown point mutations that are later identified by automated sequencing on ABIprism 310. This strategy allowed us to detect 17 different mutations located on the different exons of the CFTR gene. The most frequent was the F508del (50.74%) followed by three other mutations (G542X, W1282X and N1303K) known to be common in the Mediterranean area. For mutations (T665S, 2766 del8, F1166C, L1043R) were exclusively found, up to now, in the Tunisian population. Our results permitted to establish cystic fibrosis mutations and their distribution in Tunisia and to implement an appropriate prevention program of these diseases through the genetic council and prenatal diagnosis.

HbHope/HbS and HbS/beta-thal double compound heterozygosity in a Mauritanian family: clinical and biochemical studies.

The hemoglobin Hope was discovered in a Mauritanian family that comes from Gorgol in the southwest of the country. The family belongs to the Soninké ethnic group, which is one of the black population groups in Mauritania. Along with this abnormal hemoglobin, HbS and beta-thalassemia were also found. This family, which we refer to as D, was encountered during a survey we conducted to study hemoglobinopathies in Mauritania. First the father was identified to carry an association of HbS and HbHope, then the study was extended to the entire family members: the wife who was found to have a beta-thalassemia trait and their three children who were found to carry HbS/beta-thalassemia mutations each. All together this family carries three different mutations that resulted in a double compound heterozygosity HbHope/HbS and HbS/beta-thal.

Hemoglobin A2' (HbA2delta') in the Mauritanian population: first results of a preliminary survey.

The Mauritanian population consists of two main groups: one Arab and Berber population and one of Black African origin. The latter includes five ethnic groups: Pular, Soninke, Black Maurs, Wolof, and Bambara. Abnormal HbA2 was found in the Mauritanian population and was characterized by cellulose acetate electrophoresis at pH 8.4 followed by DNA analysis. Among 785 subjects examined, 17 were carriers of HbA2' corresponding to 2.16 percent. The highest frequency was observed among the Pular (3.09 percent), the Black Maurs (2.72 percent), the Wolof (2.27 percent), and the Soninke (2.04 percent).

Epidemiological profile of hemoglobinopathies in the Mauritanian population.

We performed a study of hemoglobin in 700 blood donors attending the Centre Hospitalier National de Nouakchott in Mauritania. We detected 116 cases of anomalies corresponding to a global prevalence of 16.6%. The analysis of the samples studied showed that 72.3 % of the donors were Blacks and 28.7 % were White Maurs ( Maures blancs). In the Black population the prevalence of hemoglobinopathies is 21.44% whereas among the White Maurs it is 4.47%. In this study, the six ethnic groups that constitute the Mauritanian population were also represented in the sample, but with different numbers. We noted some differences in the repartition of anomalies among these groups. The hemoglobin anomalies that were found are sickle cell anemia, beta-thalassemia, and delta-variant, all in heterozygous form. Geographical repartition revealed that the hemoglobin abnormalities are mostly encountered in the Southeast and the Southwestern parts of the country, which could be considered areas of high risk for hemoglobinopathies.

The erythrocyte effects of haemoglobin O(ARAB).

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 alpha-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 alpha-thalassaemia. The coexistence of -alpha/alphaalpha resulted in an average red cell density slightly greater than normal (AA) red cells. Patients heterozygous for HbOARAB with a normal complement of four alpha 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. We conclude that the erythrocytic pathogenesis of HbOARAB involves the dehydration of red cells due, at least in part, to the K:Cl cotransport system. The similarity of the charge and consequences of the presence of both HbC and HbOARAB, which are the products of mutations at opposite ends of the beta-chain, raises the possibility that this pathology is the result of a charge-dependent interaction of these haemoglobins with the red cell membrane and/or its cytoskeleton and that this abnormality is present early in red cell development.

[Contributions to the sociologic analysis of the impact of sickle cell disease on families from Northern Tunisia]. / Contribution à l'analyse sociologique des répercussions de la drépanocytose dans des familles du nord Tunisien.

The improvement of the conditions of care and quality of life of people living with drepanocytosis now constitutes a major concern of health authorities and voluntary groups (associations of sick people and their parents). In order to examine the conditions and methods of care for children living with drepanocytosis in the Bizerte region of Northern Tunisia and to understand the problems and difficulties experienced by them and their families in their daily lives, an anthropological study was carried out in the region between January 16 and February 12, 1997. Thirty-three interviews were carried out with families of infected children. The interviews were semi-directive using a pre-established protocol (interview guide). Thirty interviews were then analysed. A multitude of information concerning various medical and psycho-social aspects of drepanocytosis in the region were revealed. The data were regrouped into the following categories: circumstances of discovering the illness; explicative model of the illness as perceived by the people interviewed; conditions and methods of care of the sick child; degree of satisfaction with the health care system; psychological profile of the children interviewed; educational level of the children interviewed; repercussions the illness has on the family; impact of the illness on household budget; suggestions and comments of the people interviewed. In light of the information collected, various actions have been proposed with the goal of improving the conditions of care and quality of life of people living with drepanocytosis in the region.

Antiphospholipid antibodies: lupus anticoagulants, anticardiolipin and antiphospholipid isotypes in patients with sickle cell disease.

Antiphospholipid antibodies were investigated in 37 individuals with sickle cell disease and compared to a control group of 30 healthy subjects. Sickle cell patients included 18 homozygous sickle cell patients, 8 S/beta thalassemic patients and 11 sickle cell trait subjects. In all individuals, antiphospholipid antibodies were explored by lupus anticoagulant (LA) detection and the quantification of IgG and IgM anticardiolipin (aCL) isotypes, total antiphospholipid antibodies (APA) and IgM, IgG and IgA antiphospholipid classes. In homozygous sickle cell patients, mean level of IgG aCL and total APA were significantly increased (17.02 +/- 8.88 GPL/ml, p < 0.05 and 10.64 +/- 10.58 UPL/ml, p < 0.05 respectively). The IgG aCL, total APA and LA frequencies were 22.2%, 44.4% and 62.2%, respectively. APA isotypes were mostly IgG or IgG and IgA. In S/beta thalassemic patients, mean levels of APA were significantly increased (10.81 +/- 7.82 UPL/ml, p< 0.05). Their frequency was 71.4% and they were mostly IgG or IgG and IgA. In patients with sickle cell trait, mean levels of APA were significantly increased (10.84 +/- 5.84 UPL/ml, p < 0.01). Their frequency was 72.7% and mostly of IgG isotype. Our study showed a close association between high APA levels and sickle cell syndrome, however there was no relationship between high levels of antiphospholipid antibodies and the major complications of sickle cell disease.

Fibrinolytic response to venous occlusion in patients with homozygous sickle cell disease.

The fibrinolytic potential was evaluated in 37 patients with homozygous sickle cell disease and compared to a control group of 30 age- and sex-matched healthy volunteers. In all individuals, the euglobulin clot lysis time and plasma antigen levels of t-Pa and PAI-1 were measured before and after venous occlusion (v.o) for 10 min. The global fibrinolytic activity was normal in 4 patients (good responders to v.o), while it was decreased in 33 patients (poor responders to v.o). Among the latter, 22 patients had significantly increased baseline levels of PAI-1 Ag (82.6 +/- 27.5 ng/ml, p < 0.001) and a normal release of t-Pa Ag after v.o. In contrast, 11 patients had basal values of PAI-1 Ag comparable to those in controls with a defective release of t-Pa Ag after v.o (11.4 +/- 5.2 ng/ml, p < 0.01). These data provide evidence for reduced fibrinolytic capacity resulting from either increased basal levels of PAI-1 or defective release of t-PA.

Abnormalities of coagulation and fibrinolysis in homozygous sickle cell disease.

Abnormalities of coagulation and fibrinolysis were studied in a group of 28 children and young adults with homozygous sickle cell disease (SCD), either in the steady state (n = 12) or during painful crisis (n = 16). Coagulation was explored by standard clotting tests and by measurement of prothrombin complex factors, factor VIII (VIII:C) and antithrombin III (ATIII), protein C (PC) and protein S (PS) activities, while fibrinolytic potential was evaluated using D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) assays. In SCD patients, thrombin time (TT) was constantly shortened, both in the steady state (ratio to control 0.83 +/- 0.08, p < 0.0001) and in crisis (0.76 +/- 0.06, p < 0.0001). Mean levels of prothrombin complex were similar in asymptomatic patients to those in controls, but were significantly decreased during sickle cell crisis (p < 0.05 for factor V and p < 0.0001 for factors II, VII and X). Factor VIII:C was significantly increased, both in the steady state (207 +/- 35%, p < 0.0001) and during crisis (208 +/- 34%, p < 0.0001). PS activity was reduced int he steady state (81 +/- 12%, p < 0.01) and further diminished in crisis (68.5 +/- 27.5%, p < 0.001), while D-dimers were significantly elevated during sickle cell crisis (1028 +/- 675 ng/ml, p < 0.001). In all SCD patients, baseline levels of t-PA antigen were comparable to those in controls, whereas concentrations of PAI-1 antigen were significantly increased, either in the steady state (89.7 +/- 26.3 ng/ml, p < 0.0001) or in crisis (75.0 +/- 24.8 ng/ml, p < 0.0001). These results provide evidence for the presence of circulating activated clotting factors in SCD and for an imbalance of the profibrinolytic and antifibrinolytic systems most likely due to increased PAI-1 levels.

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.

A point mutation in the protein 4.2 gene (allele 4.2 Tozeur) associated with hereditary haemolytic anaemia.

A recessively transmitted haemolytic anaemia associated with the lack of protein 4.2 was found in a Tunisian kindred. Trace amounts of this protein (72 kD component) became visible using high-sensitivity Western blots. Band 3 and ankyrin genes were excluded as candidate genes by linkage studies, and nucleotide sequencing of band 3 cytoplasmic domain cDNA revealed no alteration. In contrast, protein 4.2 gene contained in the homozygous state a mutation at position 310: CGA-->CAA (Arg-->Gln). This mutation defining allele 4.2 Tozeur was co-inherited with the disease. The mRNA encoding the variant protein was normal in size and approximately normal in amount. Recombinant protein 4.2 Tozeur bound normally to red cell IOVs but disclosed an increased susceptibility to proteolysis in vitro. We infer that the nearly total absence of protein 4.2 in the patients results from imbalance between destruction and synthesis of mutated protein 4.2 prior to its binding to the membrane.

[Analysis of glycosylated A1c hemoglobin by liquid phase chromatography and immunoagglutination]. / Dosage de l'hémoglobine glycosylée A1c par chromatographie en phase liquide et par immunoagglutination.

Glycated hemoglobin A1c assay is performed using HPLC on cation exchange TSK SP 5 PW column. Separation is obtained in 10 minutes using graduated elution with sodium chloride and Bis-Tris buffer. Hb A1c is resolved from the other hemoglobins (F, A0 and S). Reproductibility of method is quite good and correlated with an immunologic method (DCA 2000).