Fibrinogen Mahdia: A congenitally abnormal fibrinogen characterized by defective fibrin polymerization.

INTRODUCTION: Congenital dysfibrinogenemia is a rare qualitative fibrinogen deficiency. Molecular defects that result in dysfibrinogenemia are usually caused by mutations which affect fibrinopeptide release, fibrin polymerization, fibrin cross-linking or fibrinolysis. AIM: Here, we investigated the genetic basis of hypodysfibrinogenemia in two Tunisian siblings with major bleeding. METHODS: Coagulation-related tests were performed on the patients and their family members. Functional analysis was performed in plasma fibrinogen to characterize fibrin polymerization. The sequences of fibrinogen genes were amplified and analysed by sequencing. RESULTS: Coagulation studies revealed a reduced functional and a borderline low antigenic fibrinogen plasma levels with prolonged thrombin and activated partial thromboplastin times. The fibrinogen is also characterized by a markedly impaired polymerization and could incorporate into fibrin fibres to a smaller extent (22%). Mutational screening disclosed a heterozygous single nucleotide deletion (G) at c.1025, resulting in a frameshift mutation (AαGly323GlufsX79) that is predicted to delete a part of the αC-domain containing some of the FXIII cross-linking sites. Both the normal and the aberrant Aα-chain (approximately 43 kDa) were detected by electrophoretic analysis in the patients. CONCLUSION: The new dysfunctional fibrinogen, Mahdia variant, describes its impact on fibrin assembly after the loss of the αC domains which are involved in the lateral aggregation of protofibrils. The study confirms that the truncated Aα-chain could be incorporated into mature fibrinogen molecules.

Association study of apolipoprotein E promoter polymorphism (-427 T/C) and Alzheimer's disease in a Tunisian population.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with an as yet poorly understood etiology. Both environmental and genetic factors have been implicated as predisposing factors. The apolipoprotein E (APOE) ɛ4 allele is an established genetic susceptibility factor for AD for several populations including the Tunisian population. Polymorphism rs769446 (-427 T/C) at the promoter region of the APOE gene is postulated to affect the expression of the gene through differential binding of transcription factors. AIMS: This study aims at examining the APOE promoter polymorphism rs769446 for possible association with AD in a Tunisian population. METHODS: Using a case-control study design, a sample of 85 patients and 90 controls were investigated for association with the rs769446 polymorphism. RESULTS: No evidence of association was found in this population upon comparison between patients and healthy controls or upon stratification by APOE É›4. CONCLUSIONS: Investigations of potential gene-gene and gene-environmental interactions for this polymorphism need to be further conducted.

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.

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.

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.

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.

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.

Cystic fibrosis transmembrane conductance regulator mutations and polymorphisms associated with congenital bilateral absence of vas deferens in a restricted group of patients from North Africa.

BACKGROUND: Congenital bilateral absence of vas deferens (CBAVD) is responsible for 2-6% of male infertility. It occurs in 95% of men with cystic fibrosis. This malformation is present in patients with a sterile obstructive azoospermia but without clinical evidence of cystic fibrosis. Molecular study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene responsible for cystic fibrosis could show the relationship between this disease and bilateral absence of vas deferens. PATIENTS AND METHODS: The study involved 20 male patients aged between 28-40 years, referred with suspected cystic fibrosis and in whom bilateral absence of vas deferens was confirmed by cyto-biochemical analyses and urogenital ultrasound. Molecular study of the CFTR gene was based on several techniques: DHPLC, DGGE and direct sequencing. RESULTS: Thirteen patients had CFTR mutations: F508del, G542X, W1282X, E1104X, 711+1G → T, V201M (TG) m and IVS8-5T. These mutations were associated with polymorphisms: M470V and D1270N. Seven cases presented only polymorphisms. CONCLUSION: The different mutations found in this study were associated with polymorphisms which decrease the severity of the disease and delay its onset. Thus, bilateral agenesis of the vas deferens is classed as a form of cystic fibrosis with only genital expression.

Impact of illness on mothers of children with cystic fibrosis in Tunisia: A qualitative interview study: Impact of cystic fibrosis on Tunisian mothers.

BACKGROUND: Caring for a child with cystic fibrosis (CF) is challenging and stressful and even more so in a country with limited resources. Our aim was to study the impact of CF on the daily life of mothers with children who have CF in Tunisia, emphasizing the difficulties encountered. METHODS: Overall, 20 participants were interviewed about their experiences of being caregivers and mothers of children with CF, including their knowledge about the condition, their attitude toward it, the impact of CF on their daily lives, the main difficulties they had faced, and their concerns and wishes about CF management. RESULTS: The median age of the interviewees was 39 years. None of the mothers had known about CF before; 14 of them had difficulty accepting it at first, with suicidal thoughts in one case. Six hid the illness from the close family for fear of negative prejudices. Three mothers decided to stop having children despite availability of prenatal screening. CF also influenced the mothers' social activities (n = 13) and interfered with their jobs (n = 5). It was responsible for additional expenses in all cases. Chest physiotherapy represented a daily source of coping for all participants. Hospitalization related to Pseudomonas aeruginosa infection was considered stressful and constraining by 12 mothers. The mothers' main concern was the child's life expectancy (n = 7). Their main wish was to administer a curative treatment (n = 17). CONCLUSION: CF was found to have deeply affected the mothers' psychosocial life stressing the need for the involvement of qualified psychologists and social workers together with the medical staff.

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.

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.

[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.

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.

[Calcified necrosing cystitis]. / La cystite nécrosante calcifiée.

Calcified necrotising cystitis is an urological curiosity, the authors report two cases. With subsidence of the acute process, regeneration and fibrosis take place, with contracture of the bladder and reduction of its capacity. Augmentation ileocystoplasty was performed with success in one case. The clinical and therapeutic aspects of this rare disease are discussed.

First report of molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates from a Tunisian hospital.

Fluoroquinolones are increasingly used for the treatment of infections caused by multidrug-resistant Mycobacterium tuberculosis. Our study was designed to determine the frequency of the emergence of ciprofloxacin-resistant isolates in a university hospital (Rabta University Hospital Tunis, Tunisia) and to characterize the mutations responsible for the resistance phenotype. A total of 495 clinical M. tuberculosis isolates obtained from January 2005 to July 2008 were investigated for their susceptibility to ciprofloxacin, using the standard proportion method, PCR and DNA sequencing. Four resistant isolates (0.8%) were identified. Among these, only two carried point mutations in gyrA leading to amino acid changes other than the phenotypically silent S95T substitution. No gyrB missense mutations were found in any of the clinical isolates. Although fluoroquinolone resistance is still rare in Tunisia, accurate surveillance is needed in order to control the possible emergence of resistance to fluoroquinolones, which are essential for the successful treatment of multidrug-resistant tuberculosis.