[Neonatal screening of G6PD deficiency in Tunisia]. / Depistage neonatal du deficit en G6PD en Tunisie.

The Glucose-6-phosphate dehydrogenase (G6PI) deficiency is the most common enzymopathy worldwide. WHO had classified Tunisia among countries that are moderately affected by this affection. However, no mass-screening reflecting the real incidence was realized. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis in Tunisia. A total of 1102 neonates, born in CMNT center of Maternity and of Neonatology of Tunis during the going periods from April, 2005 till May, 2005 and from June, 2006 till September, 2006, have been enclosed in the study. The samplings included 953peripheral venous blood and 149 blood cordon. Among 1102 samplings, only 976 were of use to the screening. In our mass-screening, we consider all newborns that were born in the CMNT during the period of study and were included in the screening. A dosage of the enzymatic activity was realized using spectrophotometric method. G6PD electrophoresis and molecular study by PCR/RFLP were realized for the overdrawn newborn children. Among 976 screening neonates, 43 individuals (4.4%) were found to be G6PD deficient by quantitative enzyme assay. Newborn affected were distributed in 23 boys and 20 girls (sex ratio of 1.15). The electrophoretic mobility and the molecular biology were realized for the affected newborn. Molecular characterization of 30 G6PD deficient neonates revealed that the G6PD A- was the most common and was detected in 20 of 43 individuals (66.7%), followed by G6PD Mediterranean that was detected in 6 (13.3%). At least, 4 other unknown mutations were not able to be determined by PCR/RFLP (n=4). In conclusion G6PD deficiency is frequent in our country, justifying a systematic neonatal screening, to avoid the arisen of grave consequences of this affection. The African variant is the most frequent in our country followed by the Mediterranean one.

Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Novel factor V C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding.

The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.

Familial clustering of hepatitis B virus infections and prevention of perinatal transmission by immunization with a reduced number of doses in an area of intermediate endemicity (Tunisia).

Hepatitis B surface antigen (HBsAg) was detected in 3.3% of 7162 pregnant Tunisian women tested and HBeAg in 9.6% of the HBsAg-positive mothers. Family members of 46 of these HBsAg-positive mothers (33 husbands and 61 children aged 1-6 years) were investigated for the presence of HBV markers. HBsAg was detected in 21% of the children and 18% of the husbands. Fifty children born to HBsAg-positive mothers received hepatitis B vaccine at birth, at the age of 2-3 months and at the age of 9 months. After immunization, anti-HBs were detected in 92% of them with an anti-HBs geometric mean titre of 415 mIU ml-1. Compared with the HBsAg carrier state in older siblings, the protective efficacy was estimated to be 60%. It was 100% for infants born to HBeAg-negative mothers, but only 31% for those born to HBeAg-positive mothers. For a better efficacy, the schedule of the EPI needs to be modified to include an immunization session at 1 month of age.

High interferon titer and defective NK-cell activity in the circulation of nasopharyngeal carcinoma patients.

The interferon (IFN) activity of sera from 19 patients with nasopharyngeal carcinoma (NPC) was determined by the plaque-reduction assay with vesicular stomatitis virus (VSV) in HeLa cells and compared to that of sera from matched healthy controls. High titers of interferon were detected in the sera of the NPC patients with a geometric mean titer (GMT) of 43 +/- 25 U/ml. The interferon activity of the patients' sera was acid- and heat-labile (pH = 2 and 56 degrees C for 1 hr) and could be neutralized by a goat antiserum to human IFN-gamma. Interferon titers of the patients, in contrast, to normal controls, were not correlated with natural killer (NK) activity which was abnormally low in the NPC patients. On the other hand, a high percentage of circulating cells co-expressing the LGL marker (HNK-I) and the OKT8 antigen was detected in parallel with high IFN levels in NPC patients.

Enzymatic and immunological studies of uroporphyrinogen decarboxylase in familial porphyria cutanea tarda and hepatoerythropoietic porphyria.

Uroporphyrinogen decarboxylase activity was measured in hemoglobin-free lysates from two patients with hepatoerythropoietic porphyria (HEP) and from 12 unrelated patients with familial porphyria cutanea tarda (PCT). In HEP patients, enzyme activities were 5% of normal, and familial studies clearly confirmed that patients with HEP are cases of homozygous PCT. Immunoreactive uroporphyrinogen decarboxylase was measured by developing a direct and noncompetitive enzyme immunoassay (EIA). For the 12 familial PCT patients, we found an immunoreactive protein decreased (51%) to the same extent as the catalytic activity (48%) [cross-reactive immunological material ( CRIM ) negative]. The children from the HEP family were also CRIM negative, contrasting with another HEP family previously described as CRIM positive; our data support the hypothesis of a heterogeneity in familial uroporphyrinogen decarboxylase deficiency.

Hepatitis C core antibody detection in acute hepatitis and cirrhosis patients from Tunisia.

The detection of anti-Hepatitis C virus (HCV) Core antibodies is an important addition to HCV antibody testing. In this study it appears to be more specific than the first generation HCV tests and in combination with the detection of anti-C33c antibodies, it is possibly more sensitive. In Tunisia hepatitis C virus is implicated by the presence of anti-Core antibodies in only 8% of the adult cases of acute hepatitis as opposed to 60% for Hepatitis B virus (HBV) and 4% for Hepatitis A virus (HAV). In contrast to the low prevalence of HCV infection among acute hepatitis cases, HCV infection is implicated in 35% of the cirrhosis cases. These results stress the potential importance of HCV infection in the development of cirrhosis which is a relatively common disease in North Africa.

[Hepatitis B virus infection in Tunisia]. / Infection par le virus de l'hépatite B en Tunisie.

Recent reports show that the frequency of HBsAg varies around 4 to 6% in most Mediterranean and Middle East countries. Those areas are therefore considered as areas of intermediate endemicity for hepatitis B virus (HBV) infection. The purpose of this study is to investigate the HBV global situation in Tunisia, by means of third generation testing methods. Blood samples were obtained from 3 distinct population groups from Tunis: blood donors, consisting of young male adults staff members and patients from 4 haemodialysis units patients with either acute hepatitis or liver cirrhosis They were tested for HBsAg, anti-HBs and anti-HBc by radioimmunoassay tests. HBsAg was detected in 6.5% of the young male adults, and approximately 60% had either anti-HBs or anti-HBc antibodies. Haemodialysis staff members and patients respectively displayed 9.1% and 19.5% of HBsAg positivity, but an increase of HBsAg positivity and of all HBV serum markers in relation to the amount of time spent in dialysis units was shown among the patients. After 3 years of dialysis sessions, none remained seronegative. HBsAg was detected in approximately two-thirds of the patients with acute hepatitis or liver cirrhosis, and all cirrhosis patients had at least one HBV serum marker. These global results stress the importance of HBV infection in Tunisia. Immunization against hepatitis B virus therefore has to be considered. Nevertheless, the immunization strategy must take into account the epidemiological and economic characteristics of the country.

Hb Tunis [alpha 2 beta 2 124 (H2)Pro----Ser], a new beta chain variant identified by HPLC.

During a routine hematological investigation of a child from Tunis, a silent hemoglobin variant was discovered by isoelectric-focusing. This variant was not detectable by conventional electrophoretic methods, had normal stability, expression, and oxygen affinity, and did not produce any clinical symptoms. This new variant beta 124(H2)Pro----Ser was named Hb Tunis.

Spectrin Tunis (alpha I/78): a new alpha I variant that causes asymptomatic hereditary elliptocytosis in the heterozygous state.

Spectrin Tunis (alpha 1/78) was found in the heterozygous state in a young white North-African man and his mother. Both of them presented with mild elliptocytosis. Using one-dimensional electrophoresis, a sharp 78 kd fragment was present with a reciprocal decrease of the alpha I 80 kd domain. Kinetic analysis unambiguously confirmed that the 78 kd fragment developed at the expense of the alpha I 80 domain. The alpha I 74 kd peptide was not flanked with a peptide lacking a 2 kd fragment. From this fact, it could be inferred that the site for additional proteolysis is located upstream from arginyl residue 39 and, more precisely, should lie 10 to 20 amino-acid residues (-2 kd) from the alpha-chain N-terminus. The percentage of spectrin dimers in 4 degrees C extracts was high (over 40%), contrasting with the absence of clinical symptoms related to elliptocytosis. This is the first mutation responsible for elliptocytosis found in Tunisia.

Hemoglobin Athens-Georgia [alpha 2 beta 2 40(C6)Arg----Lys] in association with beta 0-thalassemia in Tunisia.

We describe an Hb Athens-Georgia (Hb A-Ga)/beta 0-thalassemia compound heterozygosity, found in a Tunisian patient. Oxygen binding studies of red cell suspensions of this patient, containing approximately 95% Hb A-Ga, revealed an almost normal oxygen affinity. Nevertheless, dilute solutions of Hb A-Ga showed an increased overall oxygen affinity and decreased heme-heme interaction. This could be explained by a tetrameric hemoglobin with normal oxygen binding properties but with increased dissociation into monomers or dimers, as a consequence of a structural abnormality within the alpha 1 beta 2 interface. Such an interpretation would explain the increased oxygen affinity reported in previous studies performed on heterozygous Hb A/Hb A-Ga patients.

Incidence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Tunisian populations.

Screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency was performed on male students originating from several towns in Tunisia. Three hundred and twenty-five assays were made, allowing calculation of the mean value and standard deviation of G6PD (6.32 +/- 0.72 U/g Hb), the incidence of the deficiency (1.84%) and its geographic distribution in Tunisia. G6PD electrophoresis in 54 subjects showed marked predominance of the B+ type (96.2%) compared with the A+ type (1.96%). Three deficient subjects displayed an electrophotetic mobility identical to that of the A+ type of G6PD.

Presence of in vivo-activated T-cells expressing HLA-DR molecules and IL-2 receptors in peripheral blood of patients with nasopharyngeal carcinoma.

Epstein-Barr Virus (EBV) is associated with two malignant diseases, African Burkitt's Lymphoma (BL) and Undifferentiated Nasopharyngeal Carcinoma (UNPC). North Africa is a geographical area with a high incidence of NPC. Our purpose in this study was to explore cell-mediated immunity of peripheral blood lymphocytes (PBL) from patients with UNPC and DNPC. We found an elevated percentage of OKT8 cells and of large granular lymphocytes (LGL) (30-35% HNK-I-positive cells) compared to PBL from healthy matched individuals. PBL from NPC patients contained 35% HLA-DR-positive and 30% Interleukin-2 (IL-2) receptor-positive circulating lymphocytes. PBL from NPC patients exhibited a normal proliferative response to phytohemagglutinin (PHA) and Concanavalin A (Con A) and an increased response to pokeweed mitogen (PWM). Natural killer (NK) activity towards K562 cells was low in our patients who, in addition, exhibited no lytic activity against HLA-matched EBV-transformed B cells. This lack of cytotoxicity against an EBV-transformed B-cell line cannot be explained by an impairment of IL-2 secretion, and is probably a result of the presence of high numbers of OKT8 suppressor T cells.

Etiology of acute sporadic hepatitis in adults in Senegal and Tunisia.

Markers for acute hepatitis A, B, C and E virus infections were examined in the sera of 72 patients suffering from acute hepatitis in Senegal and Tunisia. Hepatitis B was responsible for 36% and hepatitis C for 21% of the cases. Acute hepatitis A was not diagnosed. HEV infection was not observed in Senegal and represents only 4% of the acute hepatitis cases in Tunisia.