Fetal Hemoglobin in Tunisian Sickle Cell Disease Patient: Relationship with Polymorphic Sequences Cis to the ß-Globin Gene.

Fetal hemoglobin (HbF) plays a dominant role in ameliorating morbidity and mortality of hemoglobinopathies. We evaluated the effects of polymorphic markers within the ß-globin gene cluster to identify the genetic mechanics that influence HbF on Tunisian sickling patients (n = 242). Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the framework polymorphism was established by PCR-sequencing, four independent regions of interest were identified: the 5' region of ß-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal (Gγ and Aγ) genes and the 5' region of ß-globin gene. The correlation of these various Haplotypes and SNPs with HbF expression and clinical data was studied. Our data showed that among the various polymorphic markers analyzed, only the sequence (AT)xN12(AT)y in LCR HS2 region was significantly associated (p < 0.05) with increased HbF levels, suggesting that the ß-globin gene cluster exerts a significant effect on HbF in sickle cell patients. This study can improve understanding of the physiopathology of the disease and aid to increase our ability to predict clinical severity.

Two new ß+ -thalassemia mutation [ß -56 (G → C); HBBc. -106 G → C] and [ß -83 (G → A); HBBc. -133 G → A] described among the Tunisian population.

OBJECTIVES: Different thalassemia mutations have been reported in various ethnic groups and geographical regions in Tunisia. In the present study, we have investigated two rare ß(+) -thalassemia mutations, that have not previously been reported in the Tunisian population [ß -56 (G > C); HBBc. -106 G > C] and [ß -83 (G > A); HBBc. -133 G > A]. METHODS: The whole ß-globin gene was directly sequenced, and haplotype analysis was conducted through a PCR/RFLP method. RESULTS: Two new mutations were identified for the first time in Tunisia. They are located within the promoter region of ß-globin gene at position -56 (G > C) and -83 (G > A). Linkage analysis using ß-globin gene cluster haplotypes showed that these two mutations were associated with Mediterranean ß-haplotype IX [- + - + + + +] and framework 2 (FW2) [CCTCT]. CONCLUSIONS: The two newly described mutations lead to the ß(+) -thalassemia among Tunisian patients. The haplotype analysis and framework assignment have helped to identify the chromosomal background associated with these mutations, and determine their origin and spread.

Haplotype map of sickle cell anemia in Tunisia.

ß-Globin haplotypes are important to establish the ethnic origin and predict the clinical development of sickle cell disease patients (SCD). To determine the chromosomal background of ß (S) Tunisian sickle cell patients, in this first study in Tunisia, we have explored four polymorphic regions of ß-globin cluster on chromosome 11. It is the 5' region of ß-LCR-HS2 site, the intervening sequence II (IVSII) region of two fetal ((G)γ and (A)γ) genes and the 5' region of ß-globin gene. The results reveal a high molecular diversity of a microsatellite configuration describing the sequences haplotypes. The linkage disequilibrium analysis showed various haplotype combinations giving 22 "extended haplotypes". These results confirm the utility of the ß-globin haplotypes for population studies and contribute to knowledge of the Tunisian gene pool, as well as establishing the role of genetic markers in physiopathology of SCD.

Gamma radiation effects on microbiological, physico-chemical and antioxidant properties of Tunisian millet (Pennisetum Glaucum L.R.Br.).

Hygienic quality of Tunisian pearl millet flour is always of major concern to consumers as well as all involved in the production, processing and distribution sectors. In the present study, the microbiological and biochemical properties of this food were examined following gamma-radiation. The D10-values for the Total Aerobic Plate Count, yeasts and moulds were respectively 1.5 and 3.7kGy. Furthermore, millet flour is commonly susceptible to mycotoxin contaminations, so the Ochratoxin A residues were also investigated; a reduction of 74% was observed with 10kGy. Moreover, the radiation process did not significantly alter fatty acids composition of the millet flour as obtained with Gas chromatography-flame ionisation detector technic. The peroxide value had increased from 26.16 to 34.43meqO2/kg with 3kGy. At 1kGy, we noticed an important loss of vitamin A of about 88.6%. In contrast, the total phenolic content, the ABTS-RSA and the DPPH-RSA of the radiated millet flour exhibited non-significant changes (p<0.05).

Microsatellite and single nucleotide polymorphisms in the ß-globin locus control region-hypersensitive Site 2: SPECIFICITY of Tunisian ßs chromosomes.

The diversity of sickle cell disease severity is attributed to several cis acting factors, among them the single nucleotide polymorphisms (SNPs) and (AT) rich region in the ß-locus control region (ß-LCR). This contains five DNase I hypersensitive sites (HS) located 6 to 22 kb upstream to the ϵ gene. The most important of these is the HS2 (5' ß-LCR-HS2), characterized by the presence of three different SNPs and a microsatellite region known to be in association with ß(S) chromosomes in various populations. The aim of this study was to present the molecular investigation of the 5' ß-LCR-HS2 site in normal and sickle cell disease individuals in order to determine if there is any correlation or specificity between these molecular markers, the ß(S) Tunisian chromosomes and phenotypical expression of sickle cell disease. One hundred and twenty-four chromosomes from Tunisian individuals (49 ß(S) carriers and 13 normal individuals) were screened by polymerase chain reaction (PCR) and sequencing for the polymorphic short tandem microsatellite repeats (AT)(X)N(12)(AT)(Y) and the three SNPs (rs7119428, rs9736333 and rs60240093) of the 5' ß-LCR-HS2. Twelve configurations of the microsatellite motif were found with an ancestral configuration elaborated by ClustalW software. Normal and mutated alleles were observed at the homozygous and heterozygous states for the three SNPs. Correlation between microsatellites and SNPs suggests that mutant SNP alleles were mainly associated, in the homozygous sickle cell disease phenotype, with the (AT)(8)N(12)GT(AT)(7) configuration, whereas, normal SNP alleles were associated with the (AT)(X)N(12)(AT)(11) configurations in normal ß(A) chromosomes. The correlation of these various configurations with Hb F expression was also investigated. The principal component analysis (PCA) showed the correlation between the homozygous sickle cell disease phenotype, mutated SNP alleles and the Benin microsatellite configuration (AT)(8)N(12)GT(AT)(7), which confirmed the specificity of this configuration to the ß(S) chromosomes. In addition, the observed high level of Hb F (14.6%) could play a protective role against Hb S to justify the modulation of sickle cell disease severity within the Benin haplotype compared to the other haplotypes. This study highlights the fact that the ß-LCR-HS2 could be a genetic marker to identify the ethnic Tunisian ß(S) chromosomes and facilitate the molecular diagnosis of sickle cell disease.