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.

A new δ chain variant, Hb A2-Tunis [δ46(CD5)Gly → Glu; HBD: c.140G>A], observed in a Tunisian family in association with a compound heterozygosity for Hb C [ß6(A3)Glu → Lys; HBB: c.19G>A] ß(0)-thalassemia [IVS-I-1 (ß143, G>A); HBB: c.92+1G>A].

We describe a new δ-globin variant, Hb A2-Tunis [δ46(CD5)Gly → Glu; HBD: c.140G>A]. This hemoglobin (Hb) variant displayed a faster electrophoretic mobility than normal Hb A2 and was expressed at 3.2%. The molecular defect was characterized by DNA sequencing analysis. Hb A2-Tunis was found in a carrier of a ß(0)-thalassemia (ß(0)-thal) [IVS I-1 (ß143, G>A); HBB: c.92 + 1G>A] and Hb C [ß6(A3)Glu → Lys; HBB: c.19G>A], presenting with a normal Hb A2 level. Phenotype and genotype investigations revealed that the patient has a total Hb A2 level of 7.1% that was expected for a ß-thalassemia (ß-thal) minor carrier.

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.

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.

Frequency and spectrum of hemochromatosis mutations in Tunisia.

The occurrence of the C282Y and H63D mutations of the HFE gene, responsible for toxic iron overload in the liver (hereditary hemochromatosis), was still unknown in Tunisia. We report the screening of 194 chromosomes from 97 randomly collected cord blood samples. The mutations were analyzed by PCR followed by DNA sequencing. The mild H63D and the severe C282Y mutations were found in 17.5+/-5.34% and 0.5+/-0.97% of the alleles, respectively. The allele frequency of the IVS 2+4 T --> C polymorphism is high (46.4+/-7.01%) in this population. Risk for homozygosity for the severe C282Y mutation is present in the Tunisian population at a low theoretical incidence. However, due to the relatively high rate of consanguinity in the country, liver pathology due to HH is not to be disregarded.

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.

Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]: a new delta chain variant detected by DNA sequencing in a Tunisian carrier of the codon 39 (C-->T) beta0-Thalassemia mutation.

We describe a new delta-globin variant, Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]. This hemoglobin (Hb) displayed an electrophoretic mobility faster than normal Hb A2 and was expressed at 2.2 %. The molecular defect was characterized by DNA sequencing and confirmed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-designed protocol. Hb A2-Pasteur-Tunis was found in a carrier of a codon 39 (C-->T) beta0-thalassemia (thal), presenting with a normal Hb A2 level. Phenotype and genotype investigations revealed that the total Hb A2 level of the patient was that expected for a minor beta-thal (4.8%).

Molecular basis of Hb H disease in southwest Iran.

Although alpha0-thalassemia (thal) defects are not very frequent in the Iranian population, Hb H disease does occur in the country. We have analyzed the alpha gene cluster of 13 patients showing the presence of Hb H to establish the molecular background of this disease in southwest Iran (Shiraz and Hormozgan provinces). Using gap-polymerase chain reaction (gap-PCR) and direct DNA sequencing we have found the --MED-I deletion, the polyadenylation signal (poly A) mutations alphaT-Saudi alpha and alphaT-Turkish alpha and Hb Constant Spring (Hb CS) in association with the common -alpha3.7 deletion. This study has revealed that: 1) at least six genotypes are responsible for Hb H disease in the area: .-alpha3.7/ --MED-I; -alpha3.7/alphaT-Saudi alpha; alphaT-Saudi alpha/alphaT-Saudi alpha; alphaCSalpha/--MED-I; --MED-I/alphaT-Turkish alpha; and the atypical forms of Hb H disease -alpha3.7/alphaCSalpha. 2) The molecular background of Hb H disease in the southwest area of Iran is more similar to the Mediterranean type than to the Southeast Asian. 3) Hb Bart's hydrops fetalis syndrome and mild, intermediate or severe postnatal Hb H disease conditions can be expected, but at a relatively low incidence. 4) The diagnostic flowchart for patients with microcytic hypochromic anemia should include iron deficiency, beta-thal, alpha+- and alpha0-thal analyses.

Rare thalassemic syndrome caused by interaction of Hb Questembert (alpha1 codon 131, TCT>CCT, Ser>Pro) with an alpha-thalassemia-2 deletion: implications for diagnosis and management.

Abnormal globin chain biosynthesis may result in deficient quantity (thalassemia) or structural variation (abnormal hemoglobins) and traditionally, they represent two phenotypically distinct groups of disorders. However, the phenotypic expression of unstable hemoglobin variants often combine features of thalassemia together with variable peripheral hemolysis. To achieve definitive diagnosis in a child presenting with hemolytic anemia along with features associated with thalassemia intermedia, we evaluated clinical, hematological, biochemical, globin biosynthetic and molecular data. Definitive diagnosis was achieved by DNA analysis which characterized the proband to be a compound heterozygote for a common alpha-thalassemia-2 deletion (3.7 kb) and Hb Questembert (alpha131[H14] Ser>Pro) caused by a C>T mutation in codon 131 of the alpha1 globin gene in trans. The phenotype of thalassemia intermedia with marked dyserythropoiesis, found in patients inheriting alpha-thalassemia mutations along with unstable alpha-globin variants (i.e., alpha-thalassemic hemoglobinopathies), represents a distinct type of thalassemic syndrome. The proband in this study additionally had variable peripheral hemolysis, presumably related to characteristics of the unstable Hb Questembert. There is minimal experience for the management of such atypical cases and this case illustrates that it is probably insufficient to monitor clinical status in patients with such hemoglobinopathies based only on the levels of hemoglobin.

Molecular spectrum of alpha-thalassemia in Tunisia: epidemiology and detection at birth.

We present the characterization of the molecular spectrum and frequency data of alpha-thal (thal) defects in Tunisia, and an evaluation of the efficacy and limitations of Hb Bart's (gamma4) measurement for the screening of alpha-thal at birth. Cord blood samples were collected from two different areas: the northeast of the country, an area where Hb H (beta4) disease frequently occurs, and Tunis, the capital city, representative of the average Tunisian population. From the first group, 110 samples with Hb Bart's and/or microcytosis at birth were selected from 1270 randomly collected samples. Two additional population samples, one from the same northeastern region (n = 90), the other from Tunis (n = 104) were collected randomly. Nine common deletional alpha-thal defects and nondeletional mutations were screened. In the northeastern samples, selected for the presence of Hb Bart's and microcytosis, the -alpha3.7 deletion was the most common defect (4.5% allele frequency) followed by a polyadenylation (poly A) signal mutation (1.8%), the five nucleotide (nt) deletion and the -alpha4.2 deletion (both 0.9%). The African polymorphism (G-->TCGGCCC at position 7238 and T-->G at 7174) was found with an allele frequency of 11% in the selected northeastern samples. In the random population samples, the overall alpha-thal allele frequency was 4% in the northeast region, against 2% in the average Tunisian population. The +14 (G-->C) polymorphism in the 5'UTR (untranslated region) of the alpha2 gene and the African polymorphism in the second intron of the same gene, were found in 3.5% of the alleles. No alpha0-thal alleles were found among the 304 blood samples studied at the DNA level during this survey.