Epidemiological and clinical characteristics of 66 Tunisian Sickle cell syndrome patients.

Introduction: Sickle cell syndrome (SCS) represent a real health problem. In this work, we propose to study the epidemiological and clinical features of 66 patients with SCS. Methods: This is a retrospective descriptive cross-sectional study carried out on a population of 66 patients with SCS, (36 S/S, 18 S/ß-thalassemia, seven S/C and five S/OArab), over a period of two years. Results: The average age of our population is 15.5 years ± 8.4. 36 patients (55%) were born to a consanguineous marriage and 35 (53%) had siblings with SCS. The average baseline hemoglobin in our patients is 9.1g/dL±1.51. S/C patients have significantly higher baseline hemoglobin than S/S, S/ß-thalassemia and S/OArab with p <0.05. Jaundice, mucosal skin pallor and hepatomegaly have been observed only in S/S, S/ß-thalassemia and S/OArab patients. The persistence of splenomegaly is more frequent in S/C than in S/S, and in S/-thalassemia than in S/S. The most common acute complications were vaso-occlusive attacks (69.7%) and worsening of anemia (54.54%). The most common chronic complication was cholelithiasis (36.36%). Conclusion: S/C patients present the best tolerated form and were the least affected by chronic complications and therefore can lead an almost normal life.

Factor V Leiden G1691A, Prothrombin G20210A, and MTHFR C677T and A1298C Mutations in Patients With Sickle Cell Disease in Tunisia.

Sickle cell disease is a genetic disorder characterized by a hypercoagulable state. Several complications in this hemoglobinopathy are increased by thrombosis. Factor V Leiden (FVL), prothrombin (PRT) G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C mutations are major inherited risk factors of thrombotic complications. In this study, our aim was to compare the frequencies of these mutations in sickle cell patients with healthy controls. The study population comprised 35 homozygous Hb S (HBB: c.20A>T) patients, 29 compound heterozygous patients [16 Hb S/ß0-thalassemia (ß0-thal), four Hb S/ß+-thal, seven Hb S/Hb C (HBB: c.19G>A) and two Hb S/Hb O-Arab (HBB: c.364G>A)] and 100 healthy subjects. All patients and controls were subjected to laboratory investigations as well as mutation genotyping. Our findings showed a severe anemia with the lowest values of protein S (PS), protein C (PC) and antithrombin (AT) in the homozygous Hb S group compared to Hb S/Hb C and Hb S/ß-thal subjects. No significant difference in FVL genotype between patients and controls was observed, whereas high frequencies of PRT G20210A, MTHFR C677T and MTHFR A1298C mutations in the Hb S patients and a significant association between the MTHFR C677T mutation and Hb S/ß0-thal were found.

Thrombophilic Mutations Among Patients with Sickle Cell Disease.

BACKGROUND: Factor V-Leiden (FVL), Prothrombin (PRT) G20210A, and Methylene Tetrahydro Folate Reductase (MTHFR) C677T and A1298C mutations are major inherited risk factors of thrombotic complications. Our aim in this study was to investigate the prevalence of these mutations among Tunisian sickle cell patients. METHODS: Study subjects comprised 64 patients and 100 healthy controls. FVL, PRT G20210A, and MTHFR genotypes were determined using a reverse dot blot based method. RESULTS: In the patient population studied, the prevalence of FV Leiden was not statistically different from controls while a significant prevalence of heterozygous PRT G20210A mutation among patients (10.93%) was found. An increased frequency of the MTHFR 677 C>T genotype was seen among patients as well as controls. The results showed no significant association between the MTHFR A1298C mutation and sickle cell disease (SCD). However, the prevalence of carrier among studied patients was 15.62% compared to 7% among healthy subjects. CONCLUSIONS: In conclusion, our data suggest a significant association between PRT G20210A and MTHFR C677T and sickle cell disease among Tunisian patients.

Determination of glucose-6-phosphate dehydrogenase cut-off values in a Tunisian population.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest enzymopathy worldwide. The incidence depends essentially on the methods used for the assessment. In this respect, we attempted in this study to set cut-off values of G6PD activity to discriminate among normal, heterozygous, and deficient individuals using the World Health Organization (WHO) classification and the receiver operating characteristics (ROC) curve analysis. METHODS: Blood samples from 250 female and 302 male subjects were enrolled in this study. The G6PD activity was determined using a quantitative assay. The common G6PD mutations in Tunisia were determined using the amplification refractory mutation system (ARMS-PCR) method. The ROC curve was used to choice the best cut-off. RESULTS: Normal G6PD values were 7.69±2.37, 7.86±2.39, and 7.51±2.35 U/g Hb for the entire, male, and female groups, respectively. Cut-off values for the total, male, and female were determined using the WHO classification and ROC curves analysis. In the male population, both cut-offs established using ROC curve analysis (4.00 U/g Hb) and the 60% level (3.82 U/g Hb), respectively are sensitive and specific resulting in a good efficiency of discrimination between deficient and normal males. For the female group the ROC cut-off (5.84 U/g Hb) seems better than the 60% level cut-off (3.88 U/g Hb) to discriminate between normal and heterozygote or homozygote women with higher Youden Index. CONCLUSIONS: The establishment of the normal values for a population is important for a better evaluation of the assay result. The ROC curve analysis is an alternative method to determine the status of patients since it correlates DNA analysis and G6PD activity.

First description of the rs45496295 polymorphism of the C/EBPE gene in ß-thalassemia intermedia patients.

The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of ß-thalassemia (ß-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of ß-thal major (ß-TM) and moderate type of ß-thal intermedia (ß-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% ß-TI patients, which was not the case in the ß-TM patients or in the control group. Thus, the T allele is consequently associated with the ß-TI group (p = 10-3). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10-3) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of ß-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.

Molecular identification of Gd A- and Gd B- G6PD deficient variants by ARMS-PCR in a Tunisian population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy. More than 200 mutations in the G6PD gene have been described. In Tunisia, the A-African and the B-Mediterranean mutations predominate the mutational spectrum. The purpose of this study was to apply the amplification refractory mutation system (ARMS-PCR) to the identification of Gd A+, Gd A- and Gd B- variants in a cohort of deficient individuals and to establish a phenotype/genotype association. 90 subjects were screened for enzymatic deficiency by spectrophotometric assay. The molecular analyses were performed in a group of 50 unrelated patients. Of the 54 altered chromosomes examined, 60% had the Gd A- mutation, 18% showed the Gd B- mutation and in 20% of cases, no mutations have been identified. The ARMS-PCR showed complete concordance with the endonuclease cleavage reference method and agreed perfectly with previous Tunisian studies where Gd A- and Gd B- were the most encountered. Also, similarities in spectrum mutations with North African and Mediterranean countries suggest gene migration from Africa to Europe through Spain. In conclusion, ARMS has been introduced in this study for common G6PD alleles identification in Tunisia. It gives some advantages compared to the traditional endonuclease digestion method since it is more convenient and timesaving and also offers the possibility to be applied in mass screening surveys.

Red cell indices: differentiation between ß-thalassemia trait and iron deficiency anemia and application to sickle-cell disease and sickle-cell thalassemia.

BACKGROUND: In Tunisia, thalassemia and sickle cell disease 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 a series of red blood cell indices and parameters in differentiation of beta-thalassemia trait (ß-TT) from iron deficiency anemia (IDA) and between homozygous sickle cell disease (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 ß-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 Youden's index 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 ß-TT, 125 IDA, 31 SS and 17 ST patients. RESULTS: Srivastava Index, mean corpuscular hemoglobin, red blood cell, Mentzer Index (MI) and mean corpuscular hemoglobin concentration show the highest reliability in discriminating ß-TT from IDA with new cut-offs slightly different from those described in literature. Ehsani Index, mean corpuscular volume, MI, Shine and Lal Index and Sirdah Index are the most powerful in the differentiation between SS and ST. CONCLUSIONS: The effectiveness and the simplicity of calculation of these indices make them acceptable and easy to use for differential diagnosis.

Red cell indices: differentiation between ß-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 ß-thalassemia trait (ß-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 ß-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 Youden's 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 ß-TT, 125 IDA, 31 SS, and 17 ST patients. RESULTS: Srivastava Index (SI) shows the highest reliability in discriminating ß-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 ß-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 ß-TT with atypical HbA2 levels.

δ0-Thalassemia in cis of ßKnossos globin gene: first homozygous description in thalassemia intermedia Libyans and first combination with codon 39 (C→T) in thalassemia intermedia Tunisian patients.

BACKGROUND: ß-Thalassemia is the most common disease among hemoglobinopathies in North African and Arab populations. In the present study we report the first description of the ß-Knossos codon27 (G→T) (ßKnossos) allele in cis with the δ059 (-A) mutation in thalassemia intermedia patients in Tunisia and Libya. METHODS: This identification was carried out by sequencing analysis of the whole coding regions of the δ- and ß-globin genes. RESULTS: We noted that heterozygous inheritance of the ßKnossos mutation results in a mild ß-thalassemia phenotype with a low level of HbA2 while homozygous leads to intermediate ß-thalassemia with an atypical high performance liquid chromatogram showing a complete absence of HbA2 and HbF. Compound heterozygosity of the ßKnossos with ß0 codon39 (C→T) is identified in a Tunisian proband for the first time and gives rise to a mild phenotype. In both families, the δ0 codon59 (-A) and the ßKnossos alleles were found to be associated with a single Mediterranean ß-haplotype I similar to that observed in previous reports from Algeria, Egypt, Cyprus, and Turkey. CONCLUSIONS: The chromosome supporting the ßKnossos and the δ0 codon59 (-A) alleles seems to be of a single Mediterranean origin. Premarital screening studies in families in which only one of the parents has typical aspects of ß-thalassemia trait and the other has a normal HbA2 level associated with abnormal red cell indices becomes a necessity to avoid missing thalassemia carriers.

Identification of candidate genes involved in clinical variability among Tunisian patients with ß-thalassemia.

The short tandem repeats (STR) are undoubtedly the most used molecular markers in genetic diversity studies. ß-thalassemia is a hereditary hemolytic anemia which is a common health problem all over the world. The disease has two main clinical forms: the severe form or ß-thalassemia major (TM) and the moderate form or ß-thalassemia intermedia (TI). To help understand this inconsistency, five STR have been studied in a group of 27 ß-thalassemic patients divided into 15 (TM) and 12 (TI). For each STR, the distribution of alleles, has been compared among patients with ß-thalassemia major (TM) and those with ß-thalassemia intermedia (TI).The comparison has shown, for the STR D14S72, one specific allele to (TM) and for the STR D14S990 and D14S68, two specific alleles to TI patients. The combination of these alleles with severe beta-thalassemia genotypes leads to the phenotype of beta-thalassemia intermedia even though there are no any attenuating factors such as XmnI Gg polymorphism at position -158 of the HBG2 promotor or the alpha-globin defects. This suggests that these alleles are associated with factors reducing ß-thalassemia clinical effects. One of this STR is located in the 5'UTR of the CEBPε gene which incodes a transcription factor expressed in the myeloid lineage. Thus, the involvement of the transcription factor CEBPε in the myeloid cells suggests that CEBPε is a candidate gene involved in the modulation of the ß-thalassemia expression through immune system reactions.

Detection of a novel splicing mutation causing analbuminemia in a Libyan family.

BACKGROUND AND OBJECTIVES: Analbuminemia is a very rare autosomal recessive disorder. It is an allelic heterogeneous defect caused by a variety of mutations within the albumin gene. We describe in this report two new cases of analbuminemia in Libyans. DESIGN AND METHODS: The 14 coding exons of the human serum albumin (HSA) gene and their intron-exon junctions were PCR amplified. The products were screened for mutations by Denaturing High Performance Liquid Chromatography (DHPLC). Samples with altered DHPLC profiles were sequenced. RESULTS: DNA sequencing revealed the presence of a novol homozygous G➔T transition in the first base of intron 11 (c.1428+1G>T), in both children. This mutation destroys the GT consensus donor sequence found at the 5' end of most intervening sequences and would cause the defective pre-mRNA splicing. CONCLUSION: Molecular diagnosis based on DHPLC and DNA sequencing represents a powerful tool to study molecular defects causing analbuminemia.

Contribution of ß-globin cluster polymorphisms to raise fetal hemoglobin levels in normal adults.

Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by continued expression of fetal hemoglobin (HbF) in adulthood. HPFH may be due not only to point mutations or large deletions in different regions of the cluster ß globin, but also to variations in several polymorphic sequences in this cluster. The objective of this work was to evaluate effects of polymorphic markers within cluster ß globin on HbF expression. For the purpose, we have explored in this first study of Tunisian HPFH four polymorphic regions of ß globin cluster in 68 healthy adults (34 subjects with high levels of HbF and 34 with normal HbF levels). Our results showed that the increase of HbF levels is associated with the -158 Gγ C â†’ T polymorphism, the TG(18)CG(2)CACG, TC TG(9)AG TG(2)CG(2) and TG(11)CG(4) configurations in the second intron of Gγ gene and the -540 ß (AT)(6)T(9) and (AT)(7)T(8) repeated sequences. Among the 34 subjects with raised levels of HbF, approximately 97% carried one or more of these six markers. This study suggests that there is a significant association between certain polymorphic configurations of the ß globin cluster and the increase of HbF levels in healthy individuals.

First description in Tunisia of a point mutation at codon 119 (CCT-->TCT) in the alpha1-globin gene: Hb Groene Hart in association with the -alpha3.7 deletion.

Herein we describe the case of a Tunisian girl who presented with 3% Hb Bart's (gamma4) 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 -alpha3.7 deletion was found in the heterozygous state. Since this finding could not explain the level of Hb Bart's at birth, or the hypochromia and microcytosis, all the alpha-globin genes were sequenced. This revealed a rare point mutation at codon 119 (CCT-->TCT) in the alpha1-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 [alpha119(H2)Pro-->Ser (alpha1)]. Here the -alpha3.7/alpha(alpha)119(CCT-->TCT) genotype is responsible for the alpha-thalassemia (thal) trait phenotype.