β-Thalassemia is a heterogeneous disorder caused by mutations that reduce or abolish the synthesis of the β-globin chain. The clinical severity of thalassemia major makes it a priority genetic disease for prevention programs involving population screening of heterozygotes and an optional prenatal diagnosis for carrier couples.
This study aimed to determine the most common β-globin gene mutations in Egypt using a real-time PCR and fluorescently labeled hybridization probes specific for each mutation and to assess the feasibility of introducing this technique in an overall thalassemia prevention program.
The study was carried out on 45 individuals: 37 β-thalassemia carriers [including five amniotic fluid (AF) samples], seven β-thalassemia major cases (including two AF samples), and one normal AF sample. The most common β-thalassemia mutations were characterized by real-time PCR with fluorescently labeled hybridization probes specific for IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39 in 28/37 (75.7%) carriers.
The most common mutation encountered was IVSI-110 (46%), followed by IVSI-1 (16.2%) and then IVSI-6 (13.5%). Codon 37 and codon 39 were not characterized in any sample. The genotype of the uncharacterized carriers was determined using a less sensitive method (reverse hybridization technique) and a relatively less common set of mutation was characterized as follows: IVSII-1(10.8%), codon 5 (5.4%), IVSII-745 (5.4%), and IVSI-116 (2.7%). The overall number of alleles detected using both techniques was calculated to be 51. The real-time PCR alone, with its assigned probes, detected 38/51(74.5%). Thirteen mutations (13/51=25.5%) remained uncharacterized by this technique (because of the unavailability of the corresponding probes). However, the reverse hybridization technique detected 48/51 alleles (94.1%). However, comparison between both techniques in terms of the shared mutations showed that the real-time PCR detected 38/38 (100%) of these mutations, whereas the reverse hybridization technique detected only 36/38 (94.7%).
Real-time PCR is a very rapid and accurate method for the detection of the β-thalassemia mutation, which may be valuable in cases for which a rapid decision has to be taken. Impediments to prenatal diagnosis as encountered in this study were attributed to refusal of termination of pregnancy by the family for religious/reasons, abortion following amniocentesis, and failure to determine the correct genotype of the AF analyzed.
Idiopathic thrombocytopenic purpura (ITP) in children is usually a self-limiting disorder. It may follow a viral infection or immunization and is caused by an inappropriate response of the immune system. Many viruses, such as parvovirus B19, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and rubella, are implicated in the occurrence of ITP.
The aim of the study was to investigate the occurrence of viral-associated ITP in Egyptian children.
Viral studies included specific immunoglobulin M for rubella, EBV, and CMV. In addition, molecular detection for parvovirus B19 was carried out.
Positive viral markers either by positive serology immunoglobulin M for rubella, CMV, or EBV or by a molecular study for parvovirus B19 were detected in 19 patients (38.8%). The most common viral infection was parvovirus B19 (30.6%), followed by EBV (16.3%), rubella (12.12%), and CMV (10.2%).
We conclude that in a large proportion of children with ITP in our region, an association with markers of acute viral infections similar to those of rubella, EBV, and CMV is present. Moreover, a significant proportion of the children had occult parvovirus B19 viremia. A study of occult viral infections is recommended in children with ITP.
β-Thalassemia major (β-TM) affects 60 000 births/year worldwide. These patients have to cope with complications of the disease that develop over time. In particular, marked hemostatic changes have been observed in patients with β-TM and β-thalassemia intermedia (β-TI).
This study aimed to determine the percentage of CD62P (P-selectin) among β-thalassemic patients and to correlate it with the presence and the presence of hemostatic abnormalities.
Our study included two groups. Group &Igr; included 45 patients with β-thalassemia, which was further subdivided into two subgroups: group Ia included 10 patients with TI and group Ib included 35 patients with TM. Group II included 15 apparently normal children as a control group, age and sex matched to the diseased group. Laboratory investigations were carried out including the following: complete blood count, serum ferritin, bleeding time, clotting time, prothrombin time and concentration, and finally CD62P (P-selectin).
The percentage of CD62P in thalassemic patients was significantly higher than that of the normal controls (P<0.001); in addition, it was higher in patients with TI than TM and in splenectomized patients than nonsplenectomized patients.
The expression of P-selectin, which is a clinical marker of platelet activation, was more prevalent in thalassemic children than the control children, especially those with β-TI, and in splenectomized patients even in the absence of thromboembolic manifestations.
Neuroblastoma (NB) is the most common malignant solid tumor in childhood and, among all childhood malignancies, is second only to leukemia. NB originates before birth in the neural crest. Tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis and the detection of TH m-RNA is very useful as a tumor marker for NB.
The aim of the present study is to detect the TH m-RNA in the peripheral blood of patients with NB by real-time reverse transcriptase-PCR (RT-PCR) at diagnosis and during therapy to determine the role of TH m-RNA in the diagnosis of NB and its relation to the treatment response.
Blood samples were collected from 32 children with advanced stages NB (stages III and IV), either newly diagnosed (n=11) or receiving treatment (n=21). Another 12 blood samples were also obtained from age-matched children with other pediatric cancers as a control group. We used real-time RT-PCR for the detection of TH m-RNA.
TH m-RNA expression was detected in peripheral blood samples obtained from five of 11 (45.4%) newly diagnosed cases and in five of 19 (26.3%) children receiving treatment. A significant difference was found between stages (III and VI) at presentation (P=0.04), LDH level (P=0.04), and bone marrow disease (P=0.025) with the detection of TH m-RNA in peripheral blood. Although no significant effect on survival outcomes had been reported in our patients, there were significant differences (P=0.04) in the response to treatment and TH m-RNA expression.
Detection of TH m-RNA by real-time RT-PCR is a reliable, quick, and easy way to detect the expression of NB cell in blood at diagnosis. Also, TH expression can be used as a tumor marker for the accurate diagnosis of NB and a predictor of treatment response.
B-cell activating factor (BAFF) plays a crucial role in B-cell development, survival, and immunoglobulin production. Excess BAFF results in the rescue of self-reactive B cells from anergy and the rescue of autoreactive T cells from the suppressing effect of dendritic cells, thus implicating a role in the development of autoimmunity.
The aim of this study was to evaluate BAFF and its receptor (BAFF-R) mRNA expression in patients with idiopathic thrombocytopenic purpura (ITP) and also to study the potential association of their expression with variations in disease severity, chronicity, and response to treatment.
Evaluation of BAFF and BAFF-R expression was carried out using a quantitative real-time PCR, in 79 ITP patients as well as 20 age-matched and sex-matched control volunteers.
The median expression level of BAFF and BAFF-R in ITP patients was significantly higher compared with that in the control group. Children had a significantly lower mean BAFF expression level compared with adults with ITP. Female patients had a significantly higher mean BAFF-R expression level compared with male patients. Patients with active ITP had a significantly higher BAFF expression compared with those in remission and those of the control group. The mean expression level of BAFF-R was significantly higher in patients with active ITP and in those in remission when it was compared with that of the control group. BAFF-R expression was significantly higher in steroid-treated patients compared with untreated patients. A significant positive correlation was found between BAFF and BAFF-R mRNA expression levels. BAFF expression was correlated positively with the median age of patients at the time of sampling and diagnosis.
Elevated BAFF expression in patients with active ITP indicates its possible role in the pathogenesis of ITP. Hence, selective antagonistic targeting of BAFF or BAFF-R in ITP patients with high levels of BAFF expression might be considered as a novel therapeutic strategy.
Increased adhesive events between endothelium and peripheral blood cells play a central role in the initiation of thromboembolic events that frequently complicate the outcome of β-thalassemia.
The aim of the study was to evaluate endothelial activation/dysfunction and activation of peripheral blood cells in children with β-thalassemia and to correlate endothelial and cellular activation markers with other hematological parameters in the same cohort, and to find out if these markers could be used as parameters that predict vascular complications in these patients.
Endothelial adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin adhesion molecule (ELAM)], and endothelin-1 (ET-1) were analyzed using immunoassays. Early activation antigen (CD69) expression on the peripheral blood leukocytes was detected using a flow cytometer in 55 β-thalassemics [30 major (β-TM); 15 intermedia (β-TI), and 10 minor] and 25 healthy age-matched and sex-matched children.
The levels of sICAM-1, sVCAM-1, ELAM, and ET-1 in β-TM and β-TI were significantly higher than those in thalassemia minor and controls. The levels of sVCAM-1 were significantly increased in splenectomized patients (P<0.001). Serum ICAM-1, sVCAM-1, and ELAM levels were positively correlated to each other in the β-TM and β-TI groups, with a significant difference (P<0.05). ET-1 was positively correlated to sVCAM-1 in the β-TM group. Ferritin was positively correlated to serum ICAM-1, VCAM-1, and ELAM in β-TM and β-TI. In β-thalassemia minor, ferritin was positively correlated only with serum ICAM-1 and VCAM-1. The expression of CD69 on leukocytes was significantly greater in β-TM, followed by β-TI and then β-thalassemia minor than the control groups. In the thalassemia minor group, CD69 expression was upregulated on monocytes, but neither on neutrophils nor on lymphocytes compared with the controls. There was no significant change in CD69 levels among splenectomized and nonsplenectomized patients. ET-1 levels were significantly correlated to CD69 expression on lymphocytes of the β-TM group.
Endothelial activation markers and activated leukocytes are significantly increased in β-thalassemics, showing that a severe degree of endothelial activation and damage along with chronic inflammation underlie the pathophysiology of vascular complications in these patients. The parameters studied might be useful markers for the follow-up of the vascular disease and may pave the way for improvements in the therapies for this disease.