Thalassemia by disruption in synthesis of alpha (?)

Thalassemia is
the most common monogenic disorder in word wild that clinically and genetically
is very heterogeneous. This disease is caused by disruption in synthesis of
alpha (?) or beta (?) globin chain which leads to reduce or absence of their
expression. The prevalence of ?-thalassemia is about 5% in general population 1,
but in countries with consanguineous marriage and malaria-risk areas including Southeast
Asia ,Mediterranean and Middle East countries such as Iran is more prevalent 2, 3.

mutations have been reported as a genetic causes of ?-thalassemia which
deletion of one or both ?-globin chain is the most one 2, 3. The
structural variants of alpha globin gene can have effect on severity of
clinical symptoms of ? and ? thalassemia, different indications in Complete
Blood Count (CBC) test and hematological phenotype 1, 4. The
genetic mechanism of these defects is often related to unequal crossing over or
nonreciprocal recombination resulting from misalignment of  homologous sequence of  alpha globin gene which lead to generate one
chromosome with single ?-globin gene deletion and another one containing three ?-globin
genes 5-7.
Two different alpha triplications in different population have been detected: 4.2
kb ?-globin gene triplication (???anti 4.2) in Asians and 3.7 kb ?-globin
gene triplication (???anti 3.7) in Africans, Middle Eastern and Mediterraneans
4, 7-9.

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The increasing
of ?:? globin chain ratio imbalance resulting in alpha triplication can lead to
the emergence of variable clinical phenotype including asymptomatic presentation,
significant anemia, ineffectual erythropoiesis and mild,  moderate or severe clinical signs in combination
with ?-globin gene deficiency 10-12. The
best description for this wide range of phenotype is that the excess alpha
chain presumably acts as a modulator to reduce the severity of disease in some patients
who have mutation in ?-globin gene, while in other cases the triplicated alpha with
deleterious effect resulted in more sever phenotype presentation 13-15.

Therefore considering
the genotype-phenotype correlation in patients with ?-triplications either
solely or in co-inheritance of ?- globin gene mutation can be very helpful for
genetic counseling and hemoglobinopathy diagnosis. There are some studies that
have been demonstrated this correlation and also the frequency of ???-gene is varied
in different population 1, 4,
6, 11, 13, 16-18. However,
there is no more inclusive study of triplicated alpha and cluster duplications in
Iranian population, hence, in order to evaluate the prevalence, clinical
symptoms and hematological parameters of ?- globin gene triplication in our
population, we considered 3900 patients who were carrier or affected for


Materials and Methods:

During the last
ten years, from 2008 to 2017, different patients from all over Iran has been
referred to Kariminejad-Najmabadi Pathology & Genetics Center with various
reasons, from unclear anemia to patients undergoing a blood transfusion. Also, alpha
and beta thalassemia carrier detection was performed for individuals who were
partner or sibling of carrier or affected individuals. Totally, three thousand
and nine hundred patients were recruited in this study.

Fresh blood
sample and complete clinical history was obtained after getting informed
consent form. Hematological parameters, Hb A2 and Hb F levels was measured by……., respectively.
Genomic DNA was extracted from 10 ml EDTA blood of each patient using salting
out method 19. 

In order to detection
of common alpha thalassemia determinants and ???anti 3.7
triplication, reverse hybridization using the alpha-Globin Strip Assay (Vienna
Lab Diagnostics GmbH) and multiplex gap-polymerase chain reaction (gap-PCR) method
were applied 11, 20, 21.
These methods were followed using Multiplex ligation-dependent
probe amplification (MLPA) for confirming the PCR results and also large
deletions or all the rearrangements identification in alpha locus.  According to manufacturer’s specifications for
SALSA MLPA KIT P140-B4 HBA (lot B4-0312) (MRC-Holland, Amsterdam, The Netherlands),
MLPA analysis was performed 22. All MLPA reaction steps including target DNA denaturation and hybridization
of MLPA probes, ligation and PCR reaction, electrophoresis for product separation
and data analysis was carried out as previous study has been done by Farashi et
al 11.

screening of ?-globin gene point mutation and deletion was done by direct Sanger
sequencing and MLPA (MRC-Holland) techniques, respectively.