genetic mutations of beta thalassemia in middle east … · 2020-01-31 · b – thalassemia...

www.wjpps.com Vol 9, Issue 2, 2020.

134

Hassan et al. World Journal of Pharmacy and Pharmaceutical Sciences

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).


138


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.


140


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.


146


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.


148


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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