Hemoglobinopathies in Iran: An Updated Review

Hemoglobinopathies are the most common single gene disorders (monogenic disorders) in the world population. Due to specific position of Iran and the presence of multi-ethnic groups in the country, there are many varieties in the molecular genetics and clinical features of hemoglobinopathies in Iran. Hemoglobinopathies include structural variants, thalassemias, and hereditary persistence of fetal hemoglobin. In this review, we look at the common structural variants in various parts of the country along with their hematological and clinical characteristics. Also, we discuss about the burden of the thalassemias in the country, different types, complications, molecular defects and therapy.


INTRODUCTION
Hemoglobinopathies, inherited disorders of hemoglobin (Hb), are public health problem in the world. Hemoglobinopathies can be divided into structural variants, the thalassemias, and hereditary persistence of fetal hemoglobin (HPFH) 1 . Taken together, they are the most common single gene disorders (monogenic disorders) in the world population 1 . Various clinical manifestations of hemoglobin disorders can be attributed to the influence of environmental factors and various genetic modifiers. Heterogenous distribution of the disease and high variation in the phenotypic manifestation of a specific mutation are the main problems with the development of programs for the control of the hemoglobinopathies 2 . In Iran, the rate of hemoglobinopathies is high that could be attributed to the medium malaria endemicity that still exist in some provinces and high rate of consanguineous marriages in the country. So, the knowledge of genetic epidemiology and clinical features of hemoglobinopathies in the Iran will be valuable in prevention programs and better diagnosis and management of Hb disorders in the country 3 .

Structural variants The β-Chain variants
Hb S as a beta chain variant results from glutamic acid → valine substitution at the 6 th codon of beta chain. This amino acid substitution in concentrated hemoglobin solutions and in the partial or fully deoxygenated conditions leads to polymerization and the occurrence of chronic hemolytic anemia and intermittent vaso-141 International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir occlusive events (sickling disorders) [4][5][6] . These events result in tissue ischemia, which leads to acute and chronic pain as well as damage of different organs in the body 7 . The low prevalence of ischemic change in some patients may be partly explained by the higher Hb F percentage among them 8 . The sickle cell anemia (SCA) patients with high Hb F level, Southern Iran, India and Eastern Saudi Arabia have the benign clinical course 9,10 .
The prevalence of sickle cell trait and SCA in southern Iran has been estimated to be 1.43 and 0.1%, respectively 11 , while in the center of Iran (Isfahan) the frequency has been reported to be 8.33% 12 .
Blood transfusion is one of the most important treatments for sickle cell disease (SCD). Transfusion slows progressive hyperplasia in bone marrow and results in reduces the risk of heart failure and face and limb changes due to bone deformation [13][14][15] . Some drugs such as hydroxyurea (HU) and 5-azacytidine by increasing formation of HbF are used in treatment the severity and the frequency of SCD episodes 16,17 . The HbS has been found to be in linkage disequilibrium with five distinct common βglobin gene cluster haplotypes are known as African haplotypes (Benin, Bantu, Senegal, and Cameroon), and Arab-Indian haplotype 18 . In Iran, genetic studies for the first time in central and southwestern Iran indicated that the β S disequilibrium gene was in linkage disequilibrium with the Arab-Indian haplotype in these regions 12,19 . The clinical presentation of SCA in southwestern Iran is associated with the elevation ratio of γ G : γ A chain and high level of Hb F in SCA patients that is related to Xmn I polymorphic site at 5´ to ε gene and is linked with Arab-Indian haplotype 20,21 . However, in western Iran, the β S gene is in linkage with the African haplotype of Benin 22 .
In 1951, another beta chain variant of hemoglobin, hemoglobin D (Hb D), was described. Variants of this Hb are Hb D-Bushman (β16 Gly→Arg), Hb D-Granada (β22 Glu→Val), Hb D-Ouled Rabah (β19 Asn→Lys), Hb D-Los Angeles or Hb D-Punjab (β121 Glu→Gln), Hb D-Iran (β22 Glu→Gln), Hb D-Ibadan (β87 Thr→Lys), ) and Hb D-Neath (β121 Glu→Ala). Only Hb D-Los Angeles and Hb D-Iran have been detected among Iranians. Hb D-Punjab was the most prevalent structural β-globin variant in Kurdish population from Western Iran and the second prevalent structural variant among Khuzestan province in Southern Iran 23,24 . Hb D in homozygous state is accounted for 95% of Hb with normal Hb F and Hb A2 levels 25 . Mild clinical presentation of Hb D-Punjab in homozygous and combined heterozygous state with β 0 -thalassemia mutation and also with α 0thalassemia mutations have been indicated 23 . In a report from South west of Iran, the combination of Hb D with β 0 thalassemia presented with a benign nature 26. Molecular genetic studies in Western Iran demonstrated an association between Hb D-Punjab mutation with haplotype I [+ ----+ +]. However, in southern Iran (Fars and Hormozgan provinces), β D alleles were linked to four haplotypes, I, V [-+ --+ + +], VII [+ -----+], and IX [-+ -+ + + +] that among them the haplotype I (67.5%) was the most prevalent 27 . In Northern Iran, (Mazandaran province) three different haplotypes were linked to Hb D-Punjab. In most cases (91.4%) β D alleles were associated with haplotype I [+----+ +] 28 .

Thalassemias
Thalassemias are divided into four types of α, β, γ and δ thalassemia. Around 1.7% of the world's populations are carriers of α-or β-thalassemia. From each 10,000 live births, approximately 4.4% of them have thalassemia 36 . In Iran, there is around 2 million thalassemia carriers 37 .
Thalassemias are more prevalent in Northern and Southern regions of the country, where the carrier rate for α-thalassemia is around 35% and for β-thalassemia is about 10% 38 .
β-Thalassemia β-thalassemia is an autosomal recessive inherited disorder due to decreased or the absence of β-globin chain production. There are 200 mutations linked with a β-thalassemia phenotype that affect the stages of β-globin gene expression and cause a reduction (β + ) or complete absence (β 0 ) of β-chain synthesis 39,40 . This hematological disorder has a high prevalence among Asian, Indian, Middle Eastern and Mediterranean populations 41 . During prenatal diagnosis (PND) programs in Iran, more than 52 thalassemic mutations with different ethnic heterogeneity have been detected 42,43 .
In three Northern provinces of Gilan, Mazandaran and Golestan, the IVS-II-1 G→A was the most prevalent (56.1%) and the CD 30 G→C (8.1%) was the second prevalent βthalassemic mutations 73 . However, in more recent study in Mazandaran and Golestan provinces of Northern Iran, the IVSII-74 (G/T) with a frequency of 54.71% was the most prevalent mutation 45 . In Northeastern province of Khorasan, the CD 8/9 +G was the most prevalent mutation (62.5%), and the second prevalent mutations were IVS-II-1 G→A, 36/37 (-T), and CD 39 C→T, each had equal frequency of 12.5% 43 . In more recent study in this province, the IVS-I-5 G→C (42.03%) was the most prevalent mutation and codon 8/9 +G had a frequency of 4.79% 46 . In Northwestern province of Tabriz, codon 36 / 37 (-T) was found to be the most prevalent mutation 44 .

Types of β-thalassemia
According to clinical manifestations, the βthalassemia is classified into three types of β-ββ-thalassemia minor (β-thal minor), β- thalassemia intermedia (β-TI) and β-thalassemia major (β-TM) 53 . β-thal minor is due to a single mutation in βgene, which leads to decrease biosynthesis of Hb A (α2β2) 54,55 . Due to the presence of excess and unmatched α chains, red blood cell (RBC) destruction increases that leads to decreased Hb level. The β-thal minor patients are asymptomatic since one β-globin gene still is normal and the clinical condition in these patients is mild-to-moderate microcytic anemia 56 . The β-thal minor patients usually experience bone pain complaint, muscle weakness, myalgia and fatigue 57 . Abnormal low plasma carnitine concentrations which lead to deficient ATP production, fatigue and bone pain complaint has been reported in these patients and carnitine and folic acid supplementation lead to a decrease in muscle weakness and bone pain complaint 58 . β-thalassemia intermedia .Genetic heterogeneity of β-TI is associated with wide clinical spectrum manifestation from mild to severe hemolytic anemia. Based on the clinical symptoms of β-TI, it can be divided into two subgroups: some patients are mildly affected with mild clinical problems until adult life. In this subgroup, Hb levels maintain between 7 and 11 g/dL and are usually rarely require blood transfusion 59 . The second subgroup consisted of patients that have severe anemia which generally present at ages 2-6 years old. These patients frequently develop clinical symptoms such as growth retardation and skeletal deformities [60][61][62] . These patients are usually diagnosed after the age of 2 years with Hb levels of 7 g/dL or free of infection and with adequate folic acid. In some carriers of this disease, normal or borderline HbA2 or isolated increased HbF is observed (up to 10%) [60][61][62] . Differential diagnosis between β-TI and β-TM is essential 63 since the first choice of β-TM management is blood transfusion, while the first step for management of patients with β-TI is usually not transfusion. In these patients, the hydroxyurea (HU) therapy, blood transfusion, and radiation therapy are therapeutic options. There are several reports indicating that erythropoietin, HU (an Hb F augmenting agent), and Minihepcidin Peptide or similar drugs (ACE-536, ACE-011), which promote RBC differentiation or maturation in the bone marrow improve anemia 64-67 . The dosage of HU which can be effective and safe in β-TI for enhancement of gamma globin chain synthesis is 8-15 mg/kg/d. In patients with β-TI, the HU therapy in combination with magnesium or Lcarnitine can be effective in improving hematologic parameters and cardiac status 68,69 . No significant association between HU response and single-nucleotide polymorphism in β-TI patients has been detected 70 . β-TM is usually diagnosed in the first 2 years of life with severe anemia, poor growth and skeletal abnormalities. Untreated β-TM usually leads to heart failure and consequently death 44 . The first step for management of patients with β-TM is blood transfusion. Blood transfusion leads to iron overload and its complications such as cardiac and liver dysfunction, immune impairment, and endocrine deficiencies 59 . Iron chelators such as deferoxamine , deferiprone, and deferasirox can reduce the excess iron in the body and prevent serious complications in patients with β-TM 71 . Deferoxamine is the standard treatment for iron overload. Because of the complications of these drugs, new studies are focused on using natural iron chelating agents [72][73][74] . So, a recent study has suggested silymarin (a flavonoid extract from the Silybum marianum) as an iron chelator could be useful 75 . Some micro RNAs (miRNAs) can regulate the maturation and the proliferation of erythroid cells, and also the expression of fetal γ-globin genes. Using miRNA for treatment of β-TM indicated a significant increase in γ-globin gene expression in the responder group 76 . However, due to high cost of health care for β-TM treatment and the lack of suitable treatment, the PND is the best way to control the prevalence of the disease. Termination of pregnancy has been allowed in Iran since 2000 in a fetus with genetic disorder 47 . Evaluating the outcome of the PND has indicated that it is an integrated primary health care approach with best infra-structure for implementing successful strategies that significantly reduced the rate of β-thalassemia 77 . Studies are now looking for novel methods with high sensitivity and specificity for detection of a paternally inherited mutation in a fetus 78 . It has been suggested that the real-time PCR high-resolution melt could be a sensitive and specific method for distinguish the paternally inherited mutation in a fetus at risk with β-TM 79 .
α-Thalassemia α-Thalassemia is a hereditary autosomal recessive disorder resulting from deletions or mutations within the α-globin gene cluster including of two alpha 1 (α1) and alpha 2 (α2) globin genes that are located on chromosome 16p13. More than 750 different variants in αglobin genes have been identified, leading to αthalassemia worldwide 80 . It is estimated that more than 5.0% of the world's population are carriers of -thalassemia 81 . The α-thalassemia is commonly found in sub-Saharan Africa, Mediterranean region, Middle East, Indian Subcontinent, East, and Southeast Asia and immigrants to these areas [82][83][84][85] . Middle East is so-called thalassemia belt. Iran is located in the Middle East between Iraq and Pakistan, and the incidence of α-thalassemia in Iran is high 50,86 .
Although the frequency of α-thalassemia carriers in Iran is not well detected, one report from Northern Iran has estimated its frequency around 15.0% 87 . In Iran, more than 19 different α-globin gene mutations have been identified, representing the heterogeneity of the population 88,89 . Common and rare mutations of α-thalassemia can be classified into deletional, and non-deletional. The most common deletional and non-deletional mutations are shown in Figure 1. Over 70 non-deletional forms of α-thalassemia have been detected that coinherited with deletional mutations (90) or with other genetic modifiers, leading to diverse genotypic and/or phenotypic expressions 91 . The spectrum of α-thalassemia mutations in different regions of Iran showed that the α -3.7 (rightward deletion), --MED (Mediterranean deletion) and α -4.2 (leftward deletion) are the major common mutations among Iranian patients 88,92 . Kerman province has the highest frequency of α 3.7 deletion among Iranian population with a frequency of around 83% 93 . However, in Gilan and Mazandaran (two Northern provinces), the frequency of α 3.7 deletion are lower than others, 42.5 and 44.9%, respectively 94 . This high prevalence of the α 3.7 deletion could be due to the high rate of consanguine marriages among Iranians 94 . The second most common mutation in other parts of Iran is different as in the Mazandaran province (Northern province) the α polyA2 is the second prevalent mutation 95 . However, in Khuzestan province (Southwest Iran) -- MED 96 , and in Hormozgan and Kermanshah provinces (Southern and Western Iran, respectively) the α 5nt 97, 98 is the second most common mutations. The presence of α-thalassemia 1 and αthalassemia 2 in trans position (--/-α) is the classic form of HbH disease known as deletional HbH disease. The α -3.7 (single deletion) and --20.5 kb and --MED (double deletions) are reported as the most deletions among Iranian HbH patients, while the α -3.7 , α -4.2 , --SEA, --MED , --THAI , --20.5 , --Tot , --FIL and --5.2 are the most observed mutations of HbH disease in different populations 89,90,99 . The most common genotype among Iranians is α 3.7 /--MED 100 .
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir In α-thalassemia carriers, the levels of mean corpuscular volume and mean corpuscular hemoglobin decreased, and the Hb A2 level was normal or slightly decreased along with normal level of Hb F 102 . Clinical severity of the of αthalassemia depends on the type of mutation (deletional or non-deletional) and the copy number of affected α-gene 103 . By timely screening, Hb Bart's hydrops fetalis (four defective α-globin genes) or Hb H disease (three defective α-globin genes) can be diagnosed during prenatal. Blood transfusion is by far the most important treatment for patients with thalassemia 4-10 , but the frequency of blood transfusion varies depending on the type of α-thalassemia. Patients with nondeletion type of Hb H disease have more symptoms at younger age and need more transfusions than patients with deletional Hb H disease 100,104,105 . In spite of the vital role of transfusion, it is associated with iron overload and adverse reactions in the recipients 106 . Adverse transfusion reactions can be divided into acute and delayed reactions, the acute reactions (more common) occurring within the first 24 hours of transfusion, and delayed reactions occurring after the first 24 hours. Hemovigilance is a set of supervision activities that is used to monitor and assess the safety of blood transfusions from donors to recipients, and the improvement of process and training of staff 107,108 . This system was introduced in Iran in 2009, which has been used in a study in Shiraz 106 .

CONCLUSION
Due to specific location of Iran and the presence of various ethnic groups in the country, there are many varieties in the molecular genetics and clinical features of hemoglobinopathies in the country. Hemoglobinopathies included structural variants, thalassemias, and HPFH. Many structural variants have been identified in Iran, but among these abnormal variants, β-globin chain variants of Hb S and Hb D and α-globin chain variants of Hb Q-Iran and Hb Setif are more common. Thalassemia is one of the major genetically inherited hematological diseases. A wide spectrum of β-thalassemia alleles has been detected among Iranians with IVSII-1 G→A as the most prevalent β-thalassemia mutation. Among Iranians, more than 19 different α-globin gene mutations have been detected, which represent the heterogeneity of the population. The α-3.7Kb was found to be the major common deletional mutation among Iranians. The first step for management of patients with severe form of thalassemia is blood transfusion; however, it leads to an iron overload and its complications. So, new therapies have recently been proposed for the disease.