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Molecular and Biochemical Study on β-Thalassemia Patients in Iraq
Update time:2013-08-09 00:52:04   【 Font: Large  Medium Small

One hundred blood samples of clinically thalassaemic patients were
collected from three thalassaemia centers in Iraq, Ibn Al-Baladi hospital, Al-
Karama hospital both in Baghdad and Hiwa hospital for cancer diseases and
thalassemia unit in Sulaymaniyah. Besides, blood samples were taken from
45 apparently healthy individuals (without Hemoglobinopathy disorders) as
a control group. Ethological distribution of the samples revealed that the
collected samples from thirteen governorates that represent various parts of
Blood samples were subjected to DNA isolation and molecular
detection of seven types of β-thalassaemia mutations (IVS1 nt.5, codon 8/9,
codon 15, codon 8, codon 30, -88, codon41/42) using a PCR -ARMS
technique. It was found that two of the diagnosed mutations were reported
for the first time within Iraqi population were codon 15 and -88 with the
frequency 7 (18.4%) and 4(10.5%), respectively and the most frequent β-
thalassaemia mutations were codon 8/9 and IVS1 nt.5 with the frequency
15 (39.5.%) and 10(26.3%), respectively. The study also reveals that codon
30 and codon 41/42 were not found within the studied samples.
The geographical distribution of positive diagnosed mutations of
thalassaemic patients observe in the map of Iraq, the results revealed the
heterogeneity of diagnosed mutations within the Iraqi regions. As can be
seen from the distribution of mutations presenting on the Iraqi map, IVS-?-5
and codon 15 were more occurred in the middle and south of Iraq, whereas
codon 8/9 and codon 8 mutations were more obvious in the middle and north
of Iraq. In addition, -88 mutation was found in the nearby cities of western
of Iraq.
levels of Serum malondialdehyde (MDA), Iron, ferritin and erythrocyte
superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities
were significantly increased in β-thalassemia patients compared with healthy
controls. A significant decrease in the levels of vitamin E and C, in addition
to the values of Total Iron Binding Capacity (T.I.B.C) were not changed in
thalassemic patients against healthy control. Ferritin levels showed a
significant positive correlation with MDA values (r2 = 0.794), while Serum
levels of vitamin E was negatively correlated with ferritin and MDA values
(r2 = 0.6815, 0.552, respectively) .
The apoptotic response shown by thalassemic cells was evaluated
using three methods, (comet assay, DNA fragmentation assay and Annexin
V-FITC(Fluorescein isothiocyanate) apoptosis). Comet assay result was
demonstrated as comet index which represent a mean of the scored which
calculate from the ratio of cell diameters (L/W) of total cells in each sample.
These results revealed that lymphocytes cells exhibited an increase apoptosis
percentage (48.2%) compare with percentage of control (6.5%). This
revealed that there was DNA damage in β-thalassemic patients.
The results obtained by DNA fragmentation assay reveal in severe
DNA damage of the thalassemic patients according to the molecular weight
of the ladder. The smear shape pattern on gel electrophoresis indicating
double strand breakage of the DNA was detected of 43.3% in studied
thalassemic samples. . Compared to the control and MW of ladder, the
thalassemic patients had higher prevalence of DNA double-strand breaks in
their leukocytes.
The final experiment of apoptosis study involved the utilization of
Annexin-V- FITC kit to monitor apoptotic and necrotic induction in
thalassemic cells. The results showed high apoptotic percentages in
thalassemia's patients (50.89%) for patients who exposed repeated
transfusion for long periods and who take irregularly chalation therapy and
they have complication due to transfusion compared to healthy people. The
percentage of apoptosis for mutations samples in thalassemic patients:
78.9% for comet assay, 76.3% for DNA fragmentation analysis and 86.8%
for labeling Annexin V assay.

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Source:University of Baghada      by AM Saud
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