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www.wjpps.com Vol 9, Issue 2, 2020.
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GENETIC MUTATIONS OF BETA THALASSEMIA IN MIDDLE EAST
COUNTRIES
Hassan Hammoud1, Ricardos Ghanem
2, Rony Abdalla
3, Pierre Semaan
4, Joëlle Azzi
5,
Edwin Parra Prada6 and Khodor Haidar Hassan
7*
1MD. Medical Lab Private Medical Lab Beirut Lebanon.
2MD, Internal Medicine Department, Beirut Arab University Bau Lebanon.
3MD. Urology Department, Beirut Arab University Bau Lebanon.
4MD Anesthesia Department, Beirut Arab University Bau.Lebanon.
5Pharmd, PhD In Health Biology (Biologie Santé) from Montpellier University – France.
Doctor At Lebanese University Faculty of Public Health 2 Branch Fanar Lebanon.
6MD. Rheumatology Department., Ospedale Sandonato. Arezzo Italy.
7MD, PhD In Health and Nutrition from Florence University Italy.
Full Professor Faculty of Public Health Lebanese University Hadath Lebanon.
ABSTRACT
Thalassemia is a type of hemolytic anemia in which appears
hemoglobin disorders depending on the location of genetic mutation
either in the gene responsible of producing alphaglobin or betaglobin
so 2 types of thalassemia were recognized as alpha or beta thalassemia
and 3 subtypes of beta thalassemia were recognized: thalassemia
minor, thalassemia intermedia and thalassemia major which is the most
severe type of beta thalassemia affecting the patient where there is
severe decrease in hemoglobin level in the blood that requires periodic
blood transfusion every 3-4 weeks to preserve sufficient hemoglobin
level in blood. It is a strict inherited disease that runs in genes from
parents to children and if one of the parents wasn’t a carrier there will
be no chances of having a thalassemic child All carries must have a
genetic testing for thalassemia of their partners before marriage. 0.13%
is the chance for the meeting of 2 carriers as couple. The study showed 4 main genetic
mutations in Beirut and its surrounding countries responsible of thalassemia, the results were
as follow: 40 patients having the mutation IVS-I-110 (G>>A), 30 patients having the
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 9, Issue 2, 134-150 Research Article ISSN 2278 – 4357
Article Received on
02 Dec. 2019,
Revised on 23 Dec. 2019,
Accepted on 12 Jan. 2020,
DOI: 10.20959/wjpps20202-15449
*Corresponding Author
Prof. Khodor Haidar
Hassan
MD, PhD In Health and
Nutrition from Florence
University Italy. Full
Professor Faculty of Public
Health Lebanese University
Hadath Lebanon.
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mutation IVS-I-1 (G>>A), 20 patients having the mutation 25bp del and 10 patients having
the mutation IVS1-6 (T>>C).
INTRODUCTION
Thalassemias are defined as an association of disorders where there is alteration of the normal
ratio of alpha to beta globin production due to a disease causing mutations in a single or
multiple genes, this abnormality in the ratio causes the unpaired chance to precipitate,
inducing by that destruction of RBC precursors in the bone marrow known as ineffective
erythropoiesis and hemolysis.
The affected patients have different degrees of anemia and extramedullary hematopoiesis so
they can be more susceptible to bone changes, impaired growth and iron overload.[1-2]
The patient usually suffers from hemolytic anemia since the first months of life and is one of
the most common genetic diseases, recall that the age of one red blood cells is 120 days so
the patient needs Blood transfusion periodically every 3 to 4 weeks depending on age and
degree of hemoglobin deficiency. In the Mediterranean area and Africa there is a group of
disorders affecting the genes responsible for the formation of hemoglobin, which in turn lead
to the imbalance in the composition of the red blood cell and thus decrease in its stability.
Types of Thalassemia
Depending on the location of defect (the gene responsible for manufacturing the protein chain
alpha or beta). Thalassemia is two types:
1 - Alpha thalassemia
2 - Beta thalassemia:
a – Thalassemia major: called Mediterranean anemia - or Cochlean anemia and is
characterized by a severe anemia within the 1st year of life
b – Thalassemia intermedia: a milder form where the symptoms manifest at the age of two
years or later.
c - Thalassemia Minor or Thalassemia trait: holder is a person who carries the trait and
inherits it for his children, it is known that there are several hundred mutations causing the
disease. There are also other types of thalassemia such as delta type.
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Causes of Thalassemia Disorder
To understand red blood cell abnormalities, we need to have an idea about the composition of
these cells, especially the genes responsible for the formation of hemoglobin chains.
Picture 1
Picture 1 Red blood cells are formed in the bone marrow and its main function is to transfer
oxygen to different body organs and to transport oxygen by binding it to hemoglobin (Figure
2-3).
Picture 2: oxygen transport by hemoglobin (bioscience.com).
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Hemoglobin Composition: Hemoglobin consists of the following parts: * iron * hem (protein)
* four pieces of protein called globin Two of these are alpha and 2 beta. (Figure 4.5).
Picture 3: Hemoglobin molecule (thalassemia.com)
Hem and globin are made inside the body by several specialized genes. The two alpha-type
globin are made up of four genes, on chromosome 16 inherited equally from both parents.
Beta-type globin is synthesized from two genes, one on chromosome 11, which is inherited
also from both parents.
Picture 4: Genetic transmission of thalassemia (thalassemia.com).
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Alpha-Thalassemia occurs if a mutation is in one of the alpha-globin genes. If the mutation is
in the genes of beta-globin, the person will have beta-thalassemia. Depending on the number
of defective genes, different types of alpha and beta thalassemia are observed.
Alpha Thalassemia
As noted above, there are four genes for alpha-globin. The symptoms of the disease are
therefore determined by the number of malformed genes:
* Silent Thalassemia - One defective gene
* Alpha Thalassemia Trait – Two defective genes
* Hemoglobin H - Three damaged genes
* Hydrops fetalis - The four genes are damaged
Beta-thalassemia
Thalassemia Minor
Also called Thalassemia trait where the affected person carries the gene Beta-Globin
composed of a normal gene and another defective one, carried do not show any symptoms of
the disease but blood changes can occur.
Thalassemia intermedia
In this case the person has a defect in both genes of Beta Globin and the resulting deficiency
is average in severity especially on the level of hemoglobin in the blood.
Thalassemia major
is one of most severe types of thalassemia, in this case the person has defect in both beta
globin genes that leads to a severe hemoglobin deficiency in the blood so that the patient
needs periodic blood transfusion Every 3 - 4 weeks to maintain a high level of hemoglobin in
the blood.
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Picture 5: Chromosome 11 defect (the thalassemia, Dr. Saad Al Ani, UAE).
Epidemiology
In the world today 250 million people have the disease trait and there are more than 300
thousand new cases yearly, the global level and available data reported by the Arab Center
for Genetic Studies indicate that Thalassemia is spread in at least 18 Arab countries,
Including the United Arab Emirates, Algeria, Bahrain, Egypt, Iraq, Jordan, Kuwait, Lebanon,
Libya, Morocco, Oman, Qatar, Saudi Arabia, Sudan, Syria, Tunisia, Palestine and Yemen, as
well as Iran. In Jordan, published statistics indicate that there are 1500 major cases and about
150,000 to 200,000 carriers of the disease. In the Arab Gulf countries, some studies indicate
that the proportion of carriers of the disease reaches up to 5% of the population, this means
the presence of a carrier among every 20 people. Studies have shown that between 5 and 6%
of the Lebanese people are carriers of thalassemia minor. The number of affected people in
Kuwait is 250-300 patients.
Picture 6: Epidemiology of thalassemia called thalassemia belt.
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The percentage of patients with thalassemia and sickle cell disease in Lebanon is between 2
and 4% of the Lebanese population, which is a high prevalence as a hereditary disease that is
transmitted only through the marriage of two people with affected genes. This means that out
of every 100 people there is about 2 carriers of the gene that do not show any symptoms of
the disease. An increase in the incidence of beta-thalassemia is noted in the following
regions: Mediterranean countries (eg Italy, Turkey, Greece and Malta), Middle East region.
(Including Iran, Iraq, Syria, Jordan, Palestine and North African countries) and includes
Egypt, Tunisia, Algeria, Morocco and some African countries.[4]
Thalassemia and heredity
Picture 7: Genetic transmission.
Table showing the types of thalassemia mutations and their distribution in the Arab countries.
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MATERIAL AND METHOD
We studied 100 patients known to be diagnosed with beta thalassemia receiving blood
transfusion on regular basis presented to several private medical laboratories for blood tests
near Beirut of Middle-eastern nationality regardless of religious and demographic distribution
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or any special habits, past medical-surgical history or family history during the period 2017-
2018
METHOD
We used the polymerase chain reaction (PCR) and allele-specific-oligonucleotide
hybridization (ASO) or restriction enzyme analysis (RE) to investigate the molecular defect
in 100 subjects diagnosed with beta-thalassemia, 20 ml of heparinized blood processed and
centrifuged to obtain a substrate of DNA.
To 0.5 mL Eppendorf tube, add 5 μL 10X reaction buffer, 5 μL nucleotide mix, 5 μL ASO
primer (either wild or mutant type primer) and 5 μL common primer, 100 ng template DNA,
and 2 U Taq polymerase, makeup to a final volume of 50 μL with sterile distilled water.
Eppendorf tubes placed on the thermal cycler to amplify the DNA by repeated cycles of
denaturation, annealing, and extension.
Following thermal cycling, electrophoresis 10 μL of the reaction sample through an agarose
gel, with a DNA size marker and stain with ethidium bromide.[3-6]
Following the last step a PCR method done for DNA amplification.
RESULTS
A sample of 100 patients with beta-thalassemia was studied to determine the genotype of the
genetic mutations responsible for the disease. The results were as follows: 40 patients with
mutation (IVS-I-110) G>A, 30 patients with mutation (IVS- I - 1) G>A, 20 patients with the
mutation of 25bp del and 10 patients carrying the mutation (IVS1 - 6) T> C.
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Clinical manifestations
1 - Pallor of the skin.
2 - Lethargy and fatigue on minimal exertion
3 - Loss of appetite.
4 - Hepatic insufficiency, liver cirrhosis, hepatomegaly and splenomegaly due to bone
marrow failure to produce red blood cells.
5 - Dysfunction of the endocrine system leading to failure in physical or sexual growth or
infertility or diabetes and delay in physical growth, such as length and weight.
6 - Changes in the bones, including the skull and the emergence of the frontal bones and the
bones of the cheeks, the nose, the upper jaw and osteoporosis because of increased iron
deposition or because of the treatment.
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7 - Increase rate of infections.
8 - Tachycardia to compensate anemia by increasing the speed of heart rate which can cause
cardiac complications over time
9 - The large number of blood transfusions may induce complications due to the
accumulation of iron in different organs of the body if the patient wasn’t on iron chelation
agent injections which helps in elimination of iron through urine and these complications
may include hepatosteatosis, darkening of the skin and a hormonal imbalance (diabetes,
hypothyroidism, hypoparathyroidism or pituitary gland hormones imbalance
10 - Cardiac problems such as cardiomegaly and arrhythmia.
11 - Higher risk of infection as a result of recurrent blood transfusion, but such diseases may
occur in rare cases because of screening tests done in all blood donation centers
12 - The negative psychological consequences for the patient due to the feeling of permanent
illness and lack of health.[5-7]
The adult patient needs also to be followed up by
1. Endocrinologist: Most patients suffer from physical and sexual growth failure, as well as
hypothyroidism, and osteoporosis either due to illness or iron chelation agent. Thalassemia
patient also suffers from diabetes due to the deposition of iron in the pancreas and needs
treatment and follow-up by specialists.
2. Cardiologist: Iron deposition may affect the myocardium and lead to serious
complications.
3. Orthopedic surgeon: To treat osteoporosis resulting from the disease.
4. Gynecologist: Most patients suffer from delayed sexual growth, as well as infertility.
Therefore, married women who wish to have children need intensive treatment for fertility
5. Gastroenterologist: Because of frequent blood transfusions, which increase the risk of
hepatitis, in addition to the effect of iron deposition within the liver and liver failure.
6. Ophthalmologist: Treatment with iron chelation agent may affect the eyesight
7. ENT Clinic: iron chelation agents treatment may affect hearing.
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Scheme 1: Pathophysiology of beta thalassemia and the resulting changes in the body,
which in turn lead to symptoms (topmedicalassistace.com).
The main lines in the treatment of thalassemia
1 - reduce the severity of the disease and prevent seizures as prevention of dehydration and
infections and protection from severe cold and hypoxia and taking antibiotics and
vaccinations
2 - blood transfusion periodically and repeatedly and throughout the life of the patient every 3
to 4 weeks, and transfusions should be done no more than six days after donation.
3. Treatment with iron chelation agents, which is a substance that attach to the iron in the
body, and enhance its excretion through the urine, which is injected through the skin for a
period of 8 - 10 hours a day parenterally or intramuscular. Iron chelation agents are effective
in elimination of iron from the body and its side effects are limited.
4 – Splenectomy: splenectomy in thalassemia has declined in the recent years. This is partly
due to a decreased prevalence of hypersplenism in adequately transfused patients. There is
also an increased appreciation of the adverse effects of splenectomy on blood coagulation. In
general, splenectomy should be avoided unless absolutely indicated.
Splenectomy is indicated in the transfusion-dependent patient when hypersplenism increases
blood transfusion requirement and prevents adequate control of body iron with chelation
therapy. An enlarged spleen—without an associated increase in transfusion requirement—is
not necessarily an indication for surgery. Patients with hypersplenism may have moderate to
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enormous splenomegaly, and some degree of neutropenia or thrombocytopenia may be
present.
Annual transfusion volume exceeding 225 to 250 mL/kg per year with packed red blood cells
(hematocrit 75 percent) may indicate the presence of hypersplenism. The volume calculation
should be corrected if the average hematocrit is less than 75 percent. The possible
development of alloantibody should also be ruled out. Splenectomy should be avoided unless
there is an inability to maintain iron balance with optimal chelation, or if there are clinically
significant complications such as pancytopenia and marked enlargement. Often,
hypersplenism develops because of a low pre-transfusion hemoglobin. Increasing the pre-
transfusion hemoglobin to between 9.5 and 10 may reverse hypersplenism.[1-9]
If a decision to perform surgery is made, partial or full splenectomy is the option. Partial
splenectomy is a complicated surgery utilized to preserve some splenic function. It should be
reserved for infants requiring splenectomy. Full splenectomy can usually be performed by
laparoscopic technique. However, open procedure is necessary in cases of marked
splenomegaly. The indications for splenectomy in hemoglobin H–Constant Spring patients
are different than in beta-thalassemia disorders. Transfusion-dependent infants with
hemoglobin H–Constant Spring respond rapidly to splenectomy but require prophylactic
anticoagulation because of a high incidence of serious thrombosis.
Patients must receive adequate immunization against Streptococcus pneumoniae,
Haemophilus influenza type B, and Neisseria meningitides prior to surgery. Splenectomy
should be avoided in children younger than five years because of a greater risk of fulminant
post-splenectomy sepsis.
After splenectomy, patients should receive oral penicillin prophylaxis (250 mg twice daily)
and be instructed to seek urgent medical attention for a fever over 101º Fahrenheit.
5 - bone marrow transplantation: This process depends on the presence of a donor, chances of
success of this process increase for those who do not suffer from iron deposition,
splenomegaly or hepatomegaly, and rejection rates are usually lower in children than adults,
it does not occur immediately after the operation, but may need 3 - 5 years after the
operation, the patient's bone marrow is first removed by giving the patient medication Which
destroys the cells of the bone marrow during 6 days in which the patient feels lethargic.
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The patient's bone marrow aspiration is done using a special syringe and under general
anesthesia. This process takes about half an hour. The process is completed by re-injecting
the healthy bone marrow into the patient. Similar to the blood transfusion process, the new
bone marrow exposes the affected bone cavity. This process has many complications and
risks.
Some centers have begun to transfer and transplant the bone marrow to the fetus during the
pregnancy instead of abortion. This process is characterized by its lower rates of rejection.
The patient is constantly followed by clinical examination and laboratory tests before each
blood transfusion. Hemoglobin level should be kept between 12, 5 - 13 g% to maintain good
growth for the child. Special vaccines / pulmonary vaccine as hemophilus influenza, hepatitis
and meningitis vaccines are also given.
WASHINGTON (Reuters) - Scientists in the United States have developed a method to treat
gene-based thalassemia using genetics, and researchers at the University of North Carolina
have devised a promising new way to treat the disease. The new treatment uses a method
called reverse DNA - a mirror image of normal DNA to prevent the affected parts of
thalassemia hemoglobin from expressing. The use of the new treatment has allowed patients
to develop normal forms of hemoglobin and researchers intend to continue their tests to know
whether the new treatment has a permanent effect or not, and intend to test on a larger
number of patients. If the gene therapy method proves success, it will give a way to cure
other similar blood diseases.[16]
Prevention
The only way to prevent thalassemia is to avoid the birth of affected newborns through:
1 - Medical consultation and medical examination before marriage, to make sure that the
partners are not carriers of thalassemia
2 - Examination of the fetus in case of suspicion of having Thalassemia to make sure and
apply the necessary medical measures
3 – Prevent when possible the marriage of relatives.
If the father is carrier of the disease, the genetic testing for the wife is mandatory. As is
known, the likelihood that a carrier of the disease joins another carrier is 0, 13%.[1-14]
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CONCLUSION
The strategy of prevention of thalassemia
1 - the establishment of special centers for Thalassemia in the provinces of the country
similar to neighboring countries as Cyprus, Greece, Italy and others to control the disease and
reduce its spread ,these centers take care of the patients and screens for carriers.
2 - Conducting a survey and identification of those who carry the disease, especially high -
risk groups like Relatives of the patients
3 - Giving genetic advice before marriage and diagnosis before birth
5 - Health education through media.
6 - Educational lectures to define what Thalassemia is, and how it is transmitted and how to
prevent it, in addition to detection of carriers and giving them advice before marriage.
Recommendations: Prevention of thalassemia: medical consultation and medical examination
before marriage are necessary to avoid thalassemia, in addition to reduction of marriage
beetwen relatives. Conducting surveys and identification of carriers of the disease,
particularly high-risk groups, like relatives of patients having thalassemia.
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