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.

Contribution of M470V variant to cystic fibrosis: First study in CF and normal Tunisian population.

PURPOSE: Determining the frequency of M470V polymorphism in cystic fibrosis and healthy cohort in Tunisia to establish the contribution of M470V polymorphism in cystic fibrosis variable presentation and course. Additionally, studying the origin of cystic fibrosis transmembrane conductance regulator gene in Tunisian population and its evolution among populations worldwide. PATIENTS AND METHODS: The genotyping of M470V marker was realized by PCR-RFLP technique in 34 unrelated patients and 50 healthy subjects. RESULTS: Statistical difference was found in the genotype and allelic distribution between CF and control groups. Exclusive association between F508del allele and M470 allele was noted. CONCLUSION: This study has contributed to better understanding involvement of the M470V polymorphism in the CF clinical expression in the Tunisian population and has confirmed the utility of this marker in the study of the origin and evolution of the CFTR locus in the human history.

Association of TGFB1 -509C/T polymorphism gene with clinical variability in cystic fibrosis patients: A case-control study.

PURPOSE: In this work, we are interested to study the implication of -509C/T polymorphism, located in the promoter region of TGFB1 (transforming growth factor ß1), in the phenotypic variability of CF patients. PATIENTS AND METHODS: The present study enrolled 111 CF patients and 100 healthy control subjects. The study of the -509C/T polymorphism was performed using PCR-RFLP method. RESULTS: We found that patients carried non-F508del homozygous mutation with TT genotype was associated to lung symptoms (P=0.04). This association was not found in the sub-groups of patients with F508del at homozygous state P=0.145. No association was found between this polymorphism and the variability of digestive, pancreatic and ileus meconial symptoms. CONCLUSION: On the basis of our results, the -509C/T polymorphism of the TGFB1 gene seems to be a modulator factor of cystic fibrosis.

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.

Preliminary study of haplotypes linked to the rare cystic fibrosis E1104X mutation.

The analysis of some extra- and intragenic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular method in clinical linkage analysis. Indeed, knowing that the molecular basis of cystic fibrosis (CF) is highly heterogeneous in our population, the study of haplotype association with normal and CF chromosomes could be very helpful in cases where one or both mutations remain unidentified. In this study, we analysed with PCR-RFLP and capillary electrophoresis some extra (pJ3.11, KM19 and XV2C) and intragenic (IVS8CA, IVS17bTA and IVS17bCA) polymorphic markers in 50 normal and 10 Tunisian patients carrying the rare E1104X mutation in order to determine the haplotype associated with this mutation. For the extragenic markers, 8 haplotypes were identified. The most frequent of them are the 221 and 112 accounting for 80% of total haplotypes. For the intragenic markers, five haplotypes were present on the E1104X chromosomes. One of them 16-31-13 accounted for 50%. To our knowledge, this is the first work to be interested to the haplotypes linked to the E1104X mutation. This preliminary study of haplotypes could be a helpful method to determine the molecular lesions responsible of this pathology.

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.

A novel 22bp deletion in a Tunisian phenylketonuria family.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase. To date, more than 530 mutations in the PAH gene have been reported. In Tunisia, this disease seems to be the result of point mutations, few studies have been published about molecular defects of PKU in our country. In this study, we report a novel deletion in exon 6 of two brothers in a Tunisian family after DHPLC analysis and sequencing of the exon 6 of the PAH gene.

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.