DETECTION OF COMMON BETA THALASSEMIA MUTATIONS AMONG EGYPTIAN PATIENTS

Beta-thalassemia is one of most common autosomal recessive disorders worldwide. Populations in the Middle- East, Mediterranean region, Central Asia, Indian and Far East countries show high prevalence for thalassemia. It is also relatively common in populations of African descent. The highest incidences are reported in Cyprus, Sardinia, and South East Asia. In Egypt, the genetic information concerning the molecular defects in β- thalassemia has not yet been fully investigated. The current study aims to detect the most common β-globin gene mutations in Egypt among β-thalassemic patients by using PCR and reverse hybridization method in an attempt to estimate the incidence of β-thalassemia mutations, a step in an assessment strategy of β-thalassemia management. Thirty seven confirmed β- thalassemia Egyptian patients were included in this study (twenty three males and fourteen females, seventeen thalassemia major and twenty thalassemia intermediate patients). Nine β-globin mutations were found in this study. IVS 1-110 was represented by 34% of the studied alleles while IVS 1-6 was represented by 23.5%, IVS 1-1 was represented by 19%, Codon 27 was represented by 6.5%, IVS 2-848 was represented by 6.5%, IVS 2-745 was represented by 2.1%, IVS 2.1 was represented by 2.5%, Codon 39 was represented by 4% and IVS 1.5 was represented by 1.5%. β-globin mutation (IVS 1- 110[G>A]) was found to be the most common homozygous mutation while, β- globin mutation (IVS 1-110[G>A]/ IVS 1- 6[T>C]) was found to be the most common heterozygous mutation. β-globin mutations (IVS 1-110[G>A], IVS 1-6[T>C] and IVS 1-1[G>A]) were found in 76% of allelic mutations. . In conclusion, establishment of β-thalassemia mutations data base may be a step in managing the diseases by carrier detection, prenatal diagnosis in high risk families, and thus can provide a tool in the prevention strategy of β-thalassemia

halassemia is a globin gene defect that results in a decreased rate of synthesis of one or more of the globin chains and a reduced rate of synthesis of the hemoglobin. Beta-thalassemia syndromes are a group of hereditary blood disorders characterized by reduced or absent beta globin chain synthesis (Bain, 2006). Beta-thalassemia is caused by the reduced (ß+) or absent (ß0) synthesis of the beta globin chains of the hemoglobin (Hb) tetramer, which is made up of two alpha globin and two beta globin chains (alpha2 beta2). Beta-thalassemia homozygotes may develop either thalassemia major or thalassemia intermediate (Olivieri and Weatherall, 2001). The decreased amount or absence of beta globin chains result in a relative excess of unbound alpha globin chains which precipitate in erythroid precursors in the bone marrow leading to their premature death and ineffective erythropoiesis. The degree of globin chain reduction is determined by the nature of the mutation at the beta globin gene (Galanello and Origa, 2010). High prevalence of beta thalassemia is present in developing countries due to the health care problems and shortage of the healthcare delivery strategy. The highest incidences of beta thalassemia are reported in populations of Mediterranean, Middle-East, Transcaucasia and Cyprus (Weatherall et al., 2007). Therefore, a prevention program for beta thalassemia in these countries is highly needed (Al-Allawi et al., 2006). As a result of Egypt position in the Mediterranean area and in the center of the Middle East, βthalassemia is considered as a major public health problem (El-Beshlawy et al., 1999). Because of the limited resources of Egypt, its healthcare system is unable to deal properly with beta thalassemia. Therefore, preventive approaches for the identification of carrier patients, genetic counseling and prenatal diagnosis are urgently needed (Ahmed et al., 2002). Many mutations had been detected which cause β-thalassemia, the information available concerning the underlying molecular defects in β-thalassemia has not yet been fully characterized (Omar et al., 2005). βthalassemia mutations varies significantly among different geographical areas, therefore the success of carrier screening and T prenatal diagnosis depends on the availability of information of prevalent mutations of such area (Edison et al., 2008).

Subjects
The studied patients were a group of attendants to the hematology clinic of Abulrish hospital, Cairo University, Egypt, suffering from β-thalassemia disease. Diagnosis of beta thalassemia was based on clinical examination, history and hematological investigations. All examination and investigations were done in accordance with the Cairo University and human Ethical Clearance Committee guidelines for clinical researches. All personal, family and medical histories of the patients were obtained with informed consent.

Methods
Patients were clinically classified into thalassemia intermediate and thalassemia major with consideration to; the age of disease onset, the age of first transfusion, frequency of blood transfusion, hepatosplenomegaly, facial and growth affection. Venous blood was withdrawn under complete aseptic conditions for CBC, retics, DNA extraction, amplification and detection of mutations by reverse hybridization technique.

β-Globin stripassay
DNA was extracted from peripheral blood leukocytes obtained from blood samples according to standard protocols and commercial kits (-Globin StripAssay MED™). Multiplex PCR amplification reaction using biotinylated primers for the extracted DNA was done according to standard protocols and commercial kits (-Globin StripAssay MED™). The amplified beta globin products were then hybridized to a test strip containing wild type and mutant oligonucleotide probes immobilized as parallel lines. Detection of the affected alleles was done by the color of the Bound biotinylated sequences according to commercial kits (-Globin StripAssay MED™).

Statistical analysis
Statistical Package for Social Sciences (SPSS) computer program (version 19 windows) was used for data analysis as follows: quantitative variables results were expressed as mean ± standard deviation (SD) or number (%). While the qualitative variables as number and percentage. P value ≤ 0.05 was considered significant and < 0.001 was considered highly significant.

RESULTS AND DISCUSSION
This study included thirty seven Egyptian patients (23 males and 14 females) who were confirmed to have βthalassemia, 17 patients with thalassemia major manifestations and 20 patients with thalassemia intermediate. Twenty three male patients present in this study accounted for 62% of the studied patients 14 thalassemia major patients and nine thalassemia intermediate patients. Fourteen female patients in this study accounted for 38% of the studied patients, 3 thalassemia major patients and 11 thalassemia intermediate patients. Regarding hematological data, when comparing thalassemia major and thalassemia intermediate groups, there were significantly higher reticulocytes, platelets and white blood cells (WBCs) and significantly lower hemoglobin (Hb), with no significant differences between patients with thalassemia major and thalassemia intermedia with regard to mean corpuscular volume and mean corpuscular hemoglobin (Table 1). El-Shanshory et al. (2014) reported that there were significantly lower red blood cells (RBCs), hemoglobin (Hb), and significantly higher reticulocytes, platelets and white blood cells (WBCs) in patients with thalassemia major compared with patients with thalassemia intermedia with no significant differences between patients with thalassemia major and thalassemia intermedia regarding mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH).
IVS 1-110(G-A) was found to be the most common homozygous mutations in six out of 14 homozygous cases (about 43%). In compound heterozygous cases, IVS 1-110(G-A) also was the most common heterozygous mutation which accounted for 13 out of 23 of the heterozygous cases representing about 56%. IVS 1-6(T-C) represented the second common homozygous mutations three out of 14 homozygous cases about 21%. In compound heterozygous cases, IVS 1-6(T-C) was the second common compound heterozygous mutation present in 11 out of 23 of the heterozygous cases accounted for 47% followed by IVS 1-1(G-A) which represented the second common homozygous mutations where three out of 14 homozygous cases about accounted for 21%. In compound heterozygous cases, IVS 1-1(G-A) occupied the third place in compound heterozygous mutations accounted for eight out of 23 of compound heterozygous cases representing about 34% (Fig.  2). These results were in agreement with those of El-Beshlawy et al. (2012) and Jiffri et al. (2010). On the other hand, El-Gawhary et al. (2007) and Elmezayen et al. (2015) reported that IVS 1-6(T-C) was more frequent than IVS 1-110(G-A).
Finally, Establishment of βthalassemia mutations data base may be an essential step in the management of the diseases by carrier detection, prenatal diagnosis in high risk families, and thus can provide a useful tool in the prevention strategy for β-thalassemia.

SUMMARY
Beta-thalassemia is one of most common autosomal recessive disorders worldwide. Populations in the Middle-East, Mediterranean region, Central Asia, Indian and Far East countries show high prevalence for thalassemia. It is also relatively common in populations of African descent. The highest incidences are reported in Cyprus, Sardinia, and South East Asia. In Egypt, the genetic information concerning the molecular defects in βthalassemia has not yet been fully investigated. The current study aims to detect the most common β-globin gene mutations in Egypt among β-thalassemic patients by using PCR and reverse hybridization method in an attempt to estimate the incidence of β-thalassemia mutations, a step in an assessment strategy of β-thalassemia management. Thirty seven confirmed βthalassemia Egyptian patients were included in this study (twenty three males and fourteen females, seventeen thalassemia major and twenty thalassemia intermediate patients). Nine β-globin mutations were found in this study. IVS 1-110 was represented by 34% of the studied alleles while IVS 1-6 was represented by 23.5%, IVS 1-1 was represented by 19%, Codon 27 was represented by 6.5%, IVS 2-848 was represented by 6.5%, IVS 2-745 was represented by 2.1%, IVS 2.1 was represented by 2.5%, Codon 39 was represented by 4% and IVS 1.5 was represented by 1.5%. β-globin mutation (IVS 1-110[G>A]) was found to be the most common homozygous mutation while, βglobin mutation (IVS 1-110[G>A]/ IVS 1-6[T>C]) was found to be the most common heterozygous mutation. β-globin mutations (IVS 1-110[G>A], IVS 1-6[T>C] and IVS 1-1[G>A]) were found in 76% of allelic mutations. . In conclusion, establishment of β-thalassemia mutations data base may be a step in managing the diseases by carrier detection, prenatal diagnosis in high risk families, and thus can provide a tool in the prevention strategy of β-thalassemia.