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.

Contribution of common CFTR variants (M470V, T854, and Q1463) to cystic fibrosis in Tunisia: haplotype analysis.

BACKGROUND & OBJECTIVES: Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR) protein, a chloride channel located in the epithelial cell membrane. Over than 2,000 CFTR mutations have been identified, which contribute to the variety of clinical phenotypes of CF. We performed a case-control study to determine p.Met470Val (M470V), p.Thr854= (T854) and p.Gln1463= (Q1463) polymorphisms frequencies in CF patients and healthy controls and to elaborate haplotype based on these SNPs. METHODS: The genotyping of M470V (exon 10), T854 (exon 14a), and Q1463 (exon 24) variants were identified using polymorphism restriction fragment length polymorphism (RFLP). RESULTS & CONCLUSION: Statistical difference was noted in the genotype distribution of two markers, M470V and T854, between CF and control groups. However, the Q1463 polymorphism is not identified in two studied groups. Three haplotypes were found in CF patients and controls. An exclusive association between the ancestral haplotype 1-1-2 and p.Phe508del (F508del) mutation was shown. In Tunisia, this is the first work to be interested in the analysis of M470V, T854 and Q1463 polymorphisms and haplotypes associated with the most common mutation, F508del, in the Tunisian population and worldwide.

Association of BglII Polymorphism in ITGA2 and (894G/T and -786T/C) Polymorphisms in eNOS Gene With Stroke Susceptibility in Tunisian Patients α2 Gene Polymorphism in α2ß1 Integrin and eNOS Gene Variants and Stroke.

BACKGROUND: This study investigated the association of BglII polymorphism in α2ß1 integrin gene (ITGA2) and eNOS (894G/T and -786T/C) polymorphisms with ischemic stroke (IS) in Tunisian patients. METHODS: The study comprised 210 patients with IS and 208 controls. The genotypes of the BglII polymorphism in ITGA2 and eNOS (894G/T and -786T/C) polymorphisms were determined using the PCR-RFLP. The χ2 test was used and the genotype data comparison included heterozygous groups. Haplotype estimation and multiple logistic regression analysis were performed to analyze the significance of polymorphisms. RESULTS: The genotype distribution of the BglII polymorphism was significantly different between cases and controls (p < 0.004). This polymorphism was associated with the risk of IS (OR = 3.38, p < 0.001) for the BglII(+/+) genotype. Likewise, the genotype distributions of eNOS (894G/T and -786T/C) polymorphisms were significantly different between the two groups (p < 0.005 and p < 0.01, respectively). The 894G/T polymorphism increased the risk of IS for the TT genotype (OR = 2.23, p < 0.008) and the GT genotype (OR = 1.74, p < 0.009). In addition, the -786T/C variant in the eNOS gene was a risk factor for IS for CC homozygous (OR = 2.52, p < 0.005). T-C Haplotype (OR = 3.06) from combination of the eNOS (894G/T and -786T/C) and T-C-BglII(+) haplotype (OR = 2.76) from combination of eNOS and ITGA2 polymorphisms represented high risks for IS. CONCLUSIONS: This study suggests that the BglII variant in ITGA2 is associated with IS susceptibility. Furthermore, the 894G/T and -786T/C polymorphisms in the eNOS gene may be considered as genetic risk factors for IS in the Tunisian population.

Glucose-6-phosphate dehydrogenase deficiency in Tunisian jaundiced neonates.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy worldwide associated with hemolysis as well as neonatal jaundice, kernicterus, and even death. The goal of this study is to determinate the prevalence of G6PD deficiency in icteric neonates and to investigate its biochemical, hematological and molecular characteristics. PATIENTS AND METHODS: This cross sectional study was carried out on 154 icteric newborns admitted to the Bechir Hamza Children's Hospital in Tunisia. Laboratory evaluations included complete blood count, total serum bilirubin level (TSB), and erythrocyte G6PD activity. The G6PD activity was determined using a quantitative assay, which allowed us to divide the total population into two groups: normal and deficient population. The common G6PD Tunisian mutations (GdA - and GdMed) were determined using the amplification refractory mutation system (ARMS-PCR) method. RESULTS: The prevalence of G6PD deficiency among total population (154 icteric newborns) was 18.83%. Male neonates showed a higher incidence of G6PD deficiency of 11.03% compared to females (7.79%). There was no statistical difference between the two groups (normal and deficient), in relation to the sex, peak TSB level, age at peak TSB, hemoglobin level, and hematocrit. However, there was a significant difference in gestational age. In the deficient group, 48.28% neonates presented the peak TSB level between 3 to 7 days and 55% of the cases show a peak TSB level greater than 250 µmol/L. The G6PD G202A variant was found in 41.37% of cases. CONCLUSION: This study shows a higher prevalence of G6PD deficiency in icteric newborns of Tunisia (18.83%). This emphasizes the necessity of neonatal screening for G6PD deficiency to prevent the exposure of these newborns to known hemolytic agents and, subsequently, to prevent kernicterus or other serious complications.

Preliminary national report on cystic fibrosis epidemiology in Tunisia: the actual state of affairs.

AIM: To establish a preliminary national report on clinical and genetic features of cystic fibrosis (CF) in Tunisian children as a first measure for a better health care organization. METHODS: All children with CF diagnosed by positive sweat tests between 1996 and 2015 in children's departments of Tunisian university hospitals were included. Data was recorded at diagnosis and during the follow-up from patients' medical records. RESULTS: In 12 departments, 123 CF children were collected. The median age at diagnosis was 5 months with a median diagnosis delay of 3 months. CF was revealed mostly by recurrent respiratory tract infections (69.9%), denutrition (55.2%), and/or chronic diarrhea (41.4%). The mean sweat chloride concentration was 110.9mmol/L. At least one mutation was found in 95 cases (77.2%). The most frequent mutations were Phe508del (n=58) and E1104X (n=15). Fifty-five patients had a Pseudomonas Aeruginosa chronic colonization at a median age of 30 months. Cirrhosis and diabetes appeared at a mean age of 5.5 and 12.5 years respectively in 4 patients each. Sixty-two patients died at a median age of 8 months. Phe508del mutation and hypotrophy were associated with death (p=0.002 and p<0.001, respectively). CONCLUSION: CF is life-shortening in Tunisia. Setting-up appropriate management is urgent.

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.

1471 delTTCT a Common Mutation of Tunisian Patients with Lysinuric Protein Intolerance.

BACKGROUND: Lysinuric protein intolerance is an inherited aminoaciduria caused by defective cationic amino acid transport. It is an autosomal recessive disease caused by mutations in the SLC7A 7 gene. The objective of this study was to identify the mutations of Tunisians patients in order to offer the genetic counseling and the prenatal diagnosis to families. METHODS: Five affected Tunisian children (4 girls and 1 boy) belonging to four consanguineous families were considered. The diagnosis was made based on the plasma for amino acids quantification by Ion Exchange chromatography, the DNA for mutational analysis by DHPLC and sequencing, and the amniotic fluid for prenatal diagnosis. RESULTS: For the 5 patients, clinical features were dominated by failure to thrive, bone marrow abnormalities, hepatosplenomegaly, and mental retardation. The diagnosis for all patients was confirmed by biochemical analysis with hyperammonemia, hyperexcretion of urinary dibasic amino acids, and a high amount of orotic acid in the urine. The 1471 delTTCT mutation was identified in exon 9 in the homozygous state for all Tunisian patients. Genetic counseling was performed for three out of four families, four heterozygous and two homozygous healthy siblings were identified. The result of prenatal diagnosis showed the presence of the 1471 de1TTCT mutation in the homozygous state for the third pregnancy and heterozygous state for the fourth. CONCLUSIONS: The 1471 deITTCT mutation seems to be a common mutation of Tunisian population. The identification of this specific mutation provides a tool for confirmatory diagnosis, genetic counseling, and prenatal 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 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.

Identification of a cystic fibrosis mutation W19X in Tunisia.

Cystic fibrosis (CF) is a common and serious condition with autosomal recessive inheritance. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The frequencies of mutations vary according to the ethnic origin of populations. We describe in this study a patient with cystic fibrosis. She was homozygous for a new nonsense mutation identified for the first time in Tunisia: W19X, which expected to cause significant morbidity. This mutation appears to be specific to Tunisian population, although, it has identified only in CF Tunisian patients. The information provided by our study contributes to defining the molecular spectrum of CF in Tunisia, to improve genetic testing and prenatal 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.

No association between an intronic polymorphism in the presenilin-1 gene and Alzheimer disease in a Tunisian population.

We examined the potential involvement of the polymorphism in intron 8 of the presenilin-1 (PSEN1) gene as a risk factor for Alzheimer disease (AD), both through independent effect and interaction with the apolipoprotein E (APOE) ε4 allele risk, in 85 patients and 90 controls. We found no significant differences in the distribution of PSEN1 genotype and allele frequency between both groups; and post stratification distribution with APOE ε4 allele. Age of onset suggests that this polymorphism influences AD progression.

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.

First report of a molecular prenatal diagnosis in a tunisian family with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI, MIM# 222700) is an inherited aminoaciduria caused by defective transport of cationic amino acids (CAAs; arginine, lysine, ornithine) at the basolateral membrane of epithelial cells in the intestine and kidney. We report the first prenatal diagnosis by direct mutational analysis of LPI performed in a Tunisian family. An amniotic fluid sample was carried out at 16 weeks of gestation in a 32-year-old Tunisian woman who consulted for prenatal diagnosis. The 1471 delTTCT mutation at homozygous state was identified indicating that the fetus was affected by LPI. The identification of this specific mutation provides a tool, which can be easily applied in Tunisia for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.

Cystic fibrosis transmembrane conductance regulator mutation spectrum in patients with cystic fibrosis in Tunisia.

AIM: To determine the frequency and types of mutations causing cystic fibrosis (CF) in Tunisia. METHODS: We analyzed the complete coding region and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 68 unrelated patients suffering from the classical form of the disease. RESULTS: Twelve different CFTR mutations accounted for 90% (123/136) of CF alleles, including F508del (47.06%), E1104X (16.18%), N1303K (6.62%), 711 + 1T > G (5.88%), W1282X (4.41%), G542X (3.67%), R1158X (1.47%), 4016insT (0.74%), and R785X (0.74%). Three novel mutations were detected in this study: I1203V (1.47%), 1811 + 5A > G (0.74%), and 4268 + 2T > G (1.47%). Fifty patients (74%) were homozygous, among which 28 (41.17%) for F508del and 10 (14.7%) for E1104X. CONCLUSIONS: Ninety-seven percent of patients were found with at least one CFTR mutation. This study contributes to a better knowledge on CF-causing mutations in different regions in Tunisia and demonstrates that a complete scanning of CFTR sequences is necessary to implement efficient programs for CF genetic screening and counseling in this part of North Africa.