Abnormal glucose tolerance in beta-thalassemia patients: possible risk factors

In beta-thalassemia major, transfusional iron overload lead to impaired glucose tolerance [IGT] then diabetes mellitus [DM]. The pathogenetic mechanisms leading from siderosis to diabetes are still poorly understood. To assess the glycometabolic status in transfusion-dependent Egyptian beta-thalassemia patients and to evaluate the possible risk factors for abnormal glucose tolerance [AGT] in them. Oral glucose tolerance test [OGTT] was done to 54 multi-transfused thalassemic patients, aged 10.1-25.1 years, and 28 age-matched normal controls with measuring serum insulin level at 0, 120 minutes. Insulin sensitivity and insulin release index were calculated according to the homeostasis model assessment [HOMA], fasting insulin glucose ratio [FIG], insulin sensitivity index for glycemia [ISl gly] and fasting insulin resistance index [FIRI]. Indicator of iron overload and liver status were recorded. Thirteen patients [24.1%] were diagnosed to have variable degrees of AGT; either DM in 6 cases [11.1%] or IGT in 7 cases [13%] and normal glucose tolerance [NGT] was in 41 patients [75.9%]. Cases with AGT had significant higher mean postprandial insulin, FIRI and HOMA insulin resistance [IR], p=0.0001 for all, and significant lower mean HOMA beta-cell, p=0.007 if compared to the cases with NGT. By stepwise logistic regression analysis, the total blood taken per year was the independent risk factor for abnormal glucose tolerance, OR=0.49, p=0.03. Serum ferritin was higher in cases with AGT compared to cases with NGT, p= 0.04 and it correlated with fasting insulin level, FIRI and HOMA IR, p=0,003,0.001, 0.001 and r=0.4, 0.5, 0.5 respectively. Chronic hepatitis C was detected in 12 patients [92.3%] with AGT. Abnormal glucose tolerance is common in multi-transfused beta-thalassemia patients, which could be attributed to impaired beta-cell function, along with insulin resistance

Molecular screening for G6PD gene mutations in children with glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase [G6PD] deficiency is a heterogeneous enzyme abnormality with a high frequency among people of African, Mediterranean and Southeast Asian origins. In almost every group studied in the Middle East, only three to four different G6PD mutations were detected. Among these, the G6PD Mediterranean mutation [563C -> T] was by far the most common. Apart from this mutation, little is known about the genetic heterogeneity of G6PD deficiency in Egypt. To screen for G6PD gene mutations in a group of Egyptian children with G6PD-deficiency who were previously screened for the Mediterranean [563C -> T] mutation. This work was conducted on twenty-one unrelated Egyptian children [17 males and 4 females] presenting with G6PD deficiency previously screened for the G6PD Mediterranean mutation [563C -> T]. Carefully-preserved DNA of patients refrigerated at -20°C and DNA of 21 age-matched normal subjects extracted from blood leukocytes by saltingout technique were screened for mutations in the G6PD gene by PCR-single strand conformation polymorphism [SSCP] analysis followed by DNA sequencing. In addition to the G6PD Mediterranean mutation 563C -> T previously identified by PCR-RFLP analysis in 6/21 male patients [28.6%], a further of 2 different mutations; G6PD A- mutation and G6PD Chatham were observed in 2/21 [9.5%] and 1/21 [4.8%] patients respectively. Twelve patients [57.1%] remained uncharacterized at the genetic level, a normal African G6PD A genotype was detected in one patient of them. Patients with G6PD Mediterranean mutation were more susceptible to hemolysis than were patients with G6PD A- and G6PD Chatham mutations. The lower prevalence of G6PD Mediterranean mutation in our patients and the finding of three different mutations in a relatively small number of G6PD-deficient subjects reflect the considerable genetic heterogeneity of G6PD deficiency of the Egyptian population

Monocytes expressing tissue factor as a diagnostic marker for neonatal sepsis

In neonatal sepsis, several clinical and laboratory parameters have been proposed for its diagnosis, however, with variable sensitivity and specificity. The bacterial products in sepsis including endotoxin induce the production of proinflammatory cytokines that evoke the expression of tissue factor [TF] on monocytes and endothelial cells. To estimate the percentage of monocytes expressing tissue factor [TF%] by flowcytometry in patients with neonatal sepsis and to delineate its significance to diagnose neonatal sepsis. Twenty-seven neonates with neonatal sepsis and positive blood culture were recruited and evaluated clinically for their risk factors. Laboratory investigations including complete blood picture, C-reactive protein [CRP] and estimation of the monocytes TF expression by flowcytometry were done. Twenty-four normal newborns were included as a control for the laboratory data. The monocytes expressing TF% of the studied patients was significantly higher than that of the controls, p-value = 0.0001. The level of TF% was significantly influenced positively by premature rupture of membrane [PROM], Multiplicity, WBC count, staff/segment ratio, CRP and negatively by gestalional age, body weight, and platelet count. The sensitivity and overall accuracy of the TF% were higher than those of the staff/segment ratio and the WBC count for diagnosing neonatal sepsis. The areas under the receiver operating characteristic curve [AUC] of TF%, staff/segment ratio and WBC count were 0.84, 0.79 and 0.60 respectively, 95% confidence interval]. The monocytes expressing TF% is a promising diagnostic and prognostic marker of infection in neonatal sepsis with high sensitivity and overall accuracy. Adding the estimation of monocytes expressing TF% to the sepsis screen may improve the diagnosis of neonatal sepsis

Correction of Aberrant pre mRNA splicing using antisense oligonucleotides in B thalassemia patients with known IVS-I,nt 110 mutation

1VS In l 110 mutation is the most common mutation in Egyptian thalassemics. The mutation causes aberrant splicing ofpre-mRNA resulting in labile RNA causing deficient B-globin chain synthesis. Antisense oligonucleotide strategy is one of the more simple technique in gene therapy. Using oligonucleotides covering the aberrant splice site gives chance for normal splicing and correction of the anomaly at the RNA level. Blocking of aberrant splicing at IVS 1nt 110 site of B-globin pre-mRNA using antisense oligonucleotides results in subsequent restoration of normal mRNA production in B-thalasseamia patients with IVS 1nt 110 mutation This study involved 10 patients with known IVS IntllO mutation; 6 homozygous and 4 heterozygous patients peripheral blood mononuclear cells were separated and duplicate liquid culture systems were set using erythropoietin and stem cell factor with and without antisense oligonucleotides [20 micro. mol/ml]. Correction of aberrant splicing was evaluated by estimation of total hemoglobin, hemoglobin F, and a reverse transcriptase polymerase chain reaction followed by agar gel electrophoresis was used to amplify and detect both aberrant mRNA and normal mRNA in duplicate samples. Five cases [50%] showed correction after antisense oligonucleotide treatment, two cases showed the appearance of normal mRNA band with absence of aberrant band and in 3 cases an increased ratio of normal mRNA band to aberrant band was found [from 2:1 to 3:1 in two cases and from 2:1 to 4:1 in the third case]. The 5 corrected cases showed significant increase in total Hb which varied between 4 to 6 folds increase. Antisense oligonucleotide treatment corrects splicing ofpre-mRNA leading to appropriate expression of B-globin mRNA which may pave the way for treatment of thalassaemia.

Genetic polymorphisms of glutathione-S-transferase and microsomal epoxide hydrolase in acquired aplastic anemia

Glutathione-S-transferase [GST] and microsomal epoxide hydrolase [mEh] are detoxifing enzymes that modulate the effects of exposure to various environmental cytotoxic and genotoxic agents, including those associated with increased risk of acquired aplastic anemia [AAA]. The GST mu1 [GSTM1], GST the ta1 [GSTT1] and mEh genes have polymorphisms with functional alteration that could explain the interindividual risks for AAA. To determine the frequency of GST and mEh polymorphisms as a risk factor for the susceptibility, clinical severity and response to treatment in a group of Egyptian paediatric patients with AAA. GST and mEh genotypes were determined by multiplex-PCR and PCR-RFLP analysis respectively in 21 patients with AAA and 20 healthy matched control subjects. The mEh enzyme activities genotypes were assessed. The incidence of the GSTT1 null genotype was significantly higher in AAA patients compared with the controls [85.7% vs. 50%] [OR 2.8, 95% CI 1.1-7.8, p=0.01]. The incidence of the heterozygous arginine [Arg] 139 polymorphism in exon 4 of the mEh gene was significantly higher in AAA patients compared with the controls [61.9% vs. 20%], [OR 3.07; 95% CI, 1.23-7.7, p=0.005]. The incidence of the fast mEh activity genotype was significantly higher in AAA patients compared with the controls [33.3% vs. 15%] [OR 2.9; 95% CI, 1.09-8.9, p=0.03]. Most patients with normal functional phenotype responded significantly favorably to treatment than patients with abnormal enzyme activity [p=0.027]. Genetic polymorphisms in biotransformation enzymes: GSTT1-null genotype, mEh His/Arg polymorphism and fast putative functional activity could be considered as risk factors to develop AAA. Moreover, abnormal functional activity of mEh enzyme was associated with worse prognosis of the disease