**4. Pathophysiology of** β**-thalassemia syndromes**

#### **4.1 Introduction**

Genetic disorders referred as the ἀ- and β-thalassemias are caused by defective hemoglobin (Hgb) chains (ἀ or β) synthesis and are mostly inherited as a Mendelian recessive [62–64]. The name of the disease" thalassemia" is derived from the Greek words: *thalassa* (sea) and *haima* (blood), implicating the geographical region where the disease was initially described due to its high prevalence. Β-thalassemia occurs mostly in people with origins near the Mediterranean Sea, Greece, Italy (Sicily, Calabria and Sardinia), Turkey, Middle East, India, Southern China, Sub Saharan Africa, south America and in the populations of Sephardic Jews and Arabs, with Cyprus (14%) and Sardinia (10,3%) having the highest carrier frequency. However, the other form of the disease, ἀ-thalassemia, is the most common among the people form the Far East, China, Vietnam, Laos, and Cambodia [63, 64].

Although considered as the rare form of the disease, it is confirmed that around 68,000 children annually are born with the various forms of thalassemia syndromes, whereas 1.5-5% of the worldwide population are considered as the carriers of these genetic abnormalities [65–68]. The high frequency of these mutations is considered as an evolutional answer to the malaria infections, providing protection

**151**

*Adaptation to Mediterranea*

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

and middle-income countries [68].

dominantly heterozygotes [62–64].

stem-cell transplantation.

**4.2 Molecular basis**

against *Plasmodium falciparum* for the genetic mutation carriers. The aberrant Hgb synthesis reduces the half-life of erythrocytes which disables completion of parasite maturation cycle [63]. Moreover, the same type of genetic aberrance has been confirmed in consanguineous marriages in some countries [64]. However, high rate of the migrations of populations caused that individuals with thalassemia-syndromes may be found in the US, Australia, Canada, South America and North Europe, making it a global health care burden [65–68]. Moreover, the general epidemiological estimation is that the prevalence of thalassemia-syndromes is about to increase, taking into the consideration the fact that infant mortality declines in low-income

Thalassemias are heterogeneous, inherited, monogenic, Hgb disorders and are initially classified as ἀ or β, depending whether genes that control ἀ- or β-globin chains synthesis are defective. This knowledge implicates that β-thalassemias occur when synthesis of the β-globin chains is reduced (β+) or absent (β-) [62–64]. Moreover, clinical, and hematological manifestations depend on how many of the genes that code β-globin synthesis are defective and whether those defects are homozygous or heterozygous. The phenotype diversity and wide range of disease

According, three culprit forms that comprise the β-thalassemia-syndromes are defined and classified by increasing severity of the symptoms: 1) β-thalassemia carrier state, also known as β-thalassemia minor, "heterozygous thalassemia" or "thalassemia trait", 2) β-thalassemia intermedia and 3) β-thalassemia mayor, also referred as "Cooley's anemia" and "Mediterranean anemia", very severe phenotype, that requires blood transfusion for survival (transfusion-dependent anemia) and has a questionable outcome. Besides these forms, there are other identified types of β-thalassemias, that are associated with various Hgb and/or clinical abnormalities or may be autosomal dominant [63, 64]. Persons with most severe forms (major) are homozygotes or compound heterozygotes for β0 or β + genes, intermedia type may be homozygotes or compound heterozygotes, while the mildest form is pre-

In the past two decades, individuals affected with β-thalassemia-syndromes are experiencing tremendous improvement in the quality of life and overall survival, due to the timely diagnosis, adequate therapy, and monitoring of the disease. However, up to date, the only cure for the disease represents allogeneic hemopoietic

The synthesis of β-globin chains in Hgb molecule physiologically is under control of two genes. Any genetic abnormality of the controlling genes, therefore, results in the absence or the reduction of the β-chain. The gene for β-chain is located in the short arm of chromosome 11, sharing the region and being arranged in the order of the development expression, with the functional genes for δ-globin, embryonic ε-globin, the fetal A-γ-globin and G-γ-globin, as well as a pseudogene (ψβ1) [63]. The molecular and clinical diversity of the β-thalassemias emerges from the data that more than 200 genetic mutations have been described up to date [63, 64, 68–70]. Accordingly, clinical, and hematological manifestation and patients' prognosis depend on the basis of imbalance of the ἀ- and β-chains synthe-

sis, therefore from the type and the extent of the genetic disturbance.

The identified and defined genetic aberrations are silent mutations (silent β-globin), mild mutations (relative reduction of β-globin) and severe mutations (complete absence of β-globin, β0) [68]. Nevertheless, these mutations are identified mostly as single-nucleotide substitutions and insertions of single nucleotides

severity lead to introduction of the concept of β-thalassemia-syndromes.

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

**3. Behcet's disease**

ocular manifestations [55, 56].

**4.1 Introduction**

Behcet's disease (BD) is an autoinflammatory and polygenic disease, more frequent in Mediterranean countries than in rest of Europe. Most cases are identified in countries of the Middle East and along the ancient Silk Route. The highest prevalence among Mediterranean countries is probably in Turkey, with estimated prevalence of 4.2/1000 in Istanbul [53]. This is a rare, sporadic, multi-systemic disease with undetermined cause. The main clinical features are constitutional symptoms and recurrent fever, oral aphthous, genital ulcers, with gastrointestinal,

Several host genetic factors are implicated in the pathogenesis of BD. The strongest is the association with the major histocompatibility complex HLA–B51 allele, which increases the risk of disease for about 6-fold. Approximately 50% of BD patients possess this HLA variant. Besides, HLA-B51 contributes to the specific clinical features in BD such as less severe disease course, but a higher frequency of

Behçet's disease can be a comorbidity of FMF, and vice versa, *MEFV* mutations are common finding in BD patients. Some *MEFV* alterations are detected more often in BD patients than healthy subjects, such as P706 polymorphism. In a cohort of Turkish patients, clinical association was found between heterozygous *MEFV*

Interestingly, arthritis in BD is self-limiting and nondestructive in nature, pointing to the existence of an inherited protective factor/s. Such a role has been observed for plasminogen activator inhibitor 1 (PAI-1), which levels were higher in synovial fluid of BD patients than healthy. PAI-1 acted protective against destructive arthritis but had promoting effect towards hyperfibrinolysis in BD vasculopathy. However, PAI-1 common polymorphism 4G/5G was not associated with

Besides, several other alterations are described to influence BD occurrence and course, including MHC class 1 polypeptide-related sequence, T cell mediated cytokine dysregulation (of IL-6, IL-8, IL-10), DNA methylation, etc. [55, 61].

Genetic disorders referred as the ἀ- and β-thalassemias are caused by defective hemoglobin (Hgb) chains (ἀ or β) synthesis and are mostly inherited as a Mendelian recessive [62–64]. The name of the disease" thalassemia" is derived from the Greek words: *thalassa* (sea) and *haima* (blood), implicating the geographical region where the disease was initially described due to its high prevalence. Β-thalassemia occurs mostly in people with origins near the Mediterranean Sea, Greece, Italy (Sicily, Calabria and Sardinia), Turkey, Middle East, India, Southern China, Sub Saharan Africa, south America and in the populations of Sephardic Jews and Arabs, with Cyprus (14%) and Sardinia (10,3%) having the highest carrier frequency. However, the other form of the disease, ἀ-thalassemia, is the most common among the people

Although considered as the rare form of the disease, it is confirmed that around

68,000 children annually are born with the various forms of thalassemia syndromes, whereas 1.5-5% of the worldwide population are considered as the carriers of these genetic abnormalities [65–68]. The high frequency of these mutations is considered as an evolutional answer to the malaria infections, providing protection

musculoskeletal, neurological, and vascular involvement [54].

mutation, principally M694V, and vascular involvement [51, 55, 57].

pathogenesis nor development of thrombosis in these patients [58–60].

**4. Pathophysiology of** β**-thalassemia syndromes**

form the Far East, China, Vietnam, Laos, and Cambodia [63, 64].

**150**

against *Plasmodium falciparum* for the genetic mutation carriers. The aberrant Hgb synthesis reduces the half-life of erythrocytes which disables completion of parasite maturation cycle [63]. Moreover, the same type of genetic aberrance has been confirmed in consanguineous marriages in some countries [64]. However, high rate of the migrations of populations caused that individuals with thalassemia-syndromes may be found in the US, Australia, Canada, South America and North Europe, making it a global health care burden [65–68]. Moreover, the general epidemiological estimation is that the prevalence of thalassemia-syndromes is about to increase, taking into the consideration the fact that infant mortality declines in low-income and middle-income countries [68].

Thalassemias are heterogeneous, inherited, monogenic, Hgb disorders and are initially classified as ἀ or β, depending whether genes that control ἀ- or β-globin chains synthesis are defective. This knowledge implicates that β-thalassemias occur when synthesis of the β-globin chains is reduced (β+) or absent (β-) [62–64]. Moreover, clinical, and hematological manifestations depend on how many of the genes that code β-globin synthesis are defective and whether those defects are homozygous or heterozygous. The phenotype diversity and wide range of disease severity lead to introduction of the concept of β-thalassemia-syndromes.

According, three culprit forms that comprise the β-thalassemia-syndromes are defined and classified by increasing severity of the symptoms: 1) β-thalassemia carrier state, also known as β-thalassemia minor, "heterozygous thalassemia" or "thalassemia trait", 2) β-thalassemia intermedia and 3) β-thalassemia mayor, also referred as "Cooley's anemia" and "Mediterranean anemia", very severe phenotype, that requires blood transfusion for survival (transfusion-dependent anemia) and has a questionable outcome. Besides these forms, there are other identified types of β-thalassemias, that are associated with various Hgb and/or clinical abnormalities or may be autosomal dominant [63, 64]. Persons with most severe forms (major) are homozygotes or compound heterozygotes for β0 or β + genes, intermedia type may be homozygotes or compound heterozygotes, while the mildest form is predominantly heterozygotes [62–64].

In the past two decades, individuals affected with β-thalassemia-syndromes are experiencing tremendous improvement in the quality of life and overall survival, due to the timely diagnosis, adequate therapy, and monitoring of the disease. However, up to date, the only cure for the disease represents allogeneic hemopoietic stem-cell transplantation.

### **4.2 Molecular basis**

The synthesis of β-globin chains in Hgb molecule physiologically is under control of two genes. Any genetic abnormality of the controlling genes, therefore, results in the absence or the reduction of the β-chain. The gene for β-chain is located in the short arm of chromosome 11, sharing the region and being arranged in the order of the development expression, with the functional genes for δ-globin, embryonic ε-globin, the fetal A-γ-globin and G-γ-globin, as well as a pseudogene (ψβ1) [63]. The molecular and clinical diversity of the β-thalassemias emerges from the data that more than 200 genetic mutations have been described up to date [63, 64, 68–70]. Accordingly, clinical, and hematological manifestation and patients' prognosis depend on the basis of imbalance of the ἀ- and β-chains synthesis, therefore from the type and the extent of the genetic disturbance.

The identified and defined genetic aberrations are silent mutations (silent β-globin), mild mutations (relative reduction of β-globin) and severe mutations (complete absence of β-globin, β0) [68]. Nevertheless, these mutations are identified mostly as single-nucleotide substitutions and insertions of single nucleotides

or small oligonucleotides causing the frameshifts in genes that code β-chains. The typical genetic abnormalities that were described are promoter mutations, being responsible for the milder phenotypes, whereas nonsense, initiation codon, splicing and frameshift mutations have been documented in more severe forms of thalassemia-syndromes, characterized with the complete absence of β-chains [62, 63]. Deletions of the gene are randomly identified aberrations, where the deletional removal of one or several genes from the chromosome 11 causes very rare forms of thalassemias, designated as δβ-, γδβ- and εγδβ-thalassemia [62].

An autosomal recessive pattern of thalassemia inheritance implicates that both parents have to be heterozygotes, owing a copy of a β-globin gene mutation. Possible outcomes in the affected family may be that every child has: 1) 25% chance of being affected, 2) 50% of being an symptom free and carrier, and 3) 25% of not being affected nor a carrier [63, 64].

#### **4.3 Genetic modifiers**

Pathophysiological perception why individuals with beta-thalassemia syndromes may clinically appear very heterogeneous, is based on the perseverance of three group of factors that may modify the disease. These factors are designated as genetic modifiers and are explained as genetic variants that induce differences in disease phenotype [64]. Genetic variants that may impact the imbalance of globin chains are categorized as primary modifiers. The other pathogenetic factors that may alleviate the severity of β-thalassemia major are: coinheritance of an ἀ-thalassemia gene and fetal Hgb production, within the β-globin cluster and are classified as secondary modifiers [62, 68].

Coexistence of ἀ-thalassemia enables decreased ἀ-globin chain synthesis, therefore significantly reduces imbalance between the ἀ/non-ἀ-chain in erythrocytes [63]. Increased γ-chain synthesis, in adult life, encounters the excess of ἀ-chains, therefore enables the survival of the erythrocytes that contain fetal hemoglobin, marked as HbF cells. It may be that deletion mutation or point mutation within the β-globin gene cluster simultaneously trigger a rise in fetal Hgb production [62]. According to some research, the increase of HbF synthesis indicates a single nucleotide polymorphism in one of the γ-globin gene promoters or somewhere in the globin locus, resulting in the overexpression of the related gene [62]. It was reported that HbF, that is highly predominant in individuals with severe forms of thalassemia, may account for their improved survival [71]. Moreover, the inverse correlation of HbF levels and factors that reflect disease morbidity was observed, so as the finding that milder phenotypes present with the increased numbers of HbF cells [62, 72]. In addition to this knowledge, it was suggested that certain therapeutic treatments (hydroxyurea) may induce the production of HbF, hence produce less of a need for blood transfusion [73].

Tertiary modifiers are recognized to be genetic and environmental factors that modulate disease complication rates. The results of the molecular studies revealed genetic polymorphisms as possible pathogenetic factors involved in cardiac iron overload, hyperbilirubinemia, and Gilbert disease, osteoporosis, and infections susceptibility, that occur in patients with β-thalassemia syndromes [63, 68, 74–76].

#### **4.4 Pathophysiology**

Essential pathophysiological determinant in β-thalassemia syndromes is the uncoupling of the synthesis of the ἀ- and the β-chain, where β-chain synthesis is reduced or absent, resulting in the accumulation of ἀ-globin tetramers in the erythroid precursors [62–64]. This phenomenon eventually leads to an ineffective

**153**

damage [83, 85].

hemochromatosis [62].

*Adaptation to Mediterranea*

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

such as venous thrombosis [63, 64, 68, 79].

to the hepcidin downregulation and its deficiency [62, 63].

erythropoiesis, that is a key feature responsible for various pathophysiological consequences during the course of the disease. Erythrocytes and its precursors (mostly polychromatophilic erythroblasts) are filled with precipitated ἀ-globin tetramers, forming inclusive bodies, causing oxidative membrane damage and subsequent apoptosis [62–64, 77]. Physiologically, biochemical detoxification would be efficient to eliminate harmful proteins from the affected cells. Nevertheless, in the

Premature erythroid cell death in the bone marrow (ineffective erythropoiesis) and in the peripheral blood (hemolysis) cause chronic microcytic-hypochromic hemolytic anemia, that is a persistent finding in persons with thalassemia. Interestingly, hemolysis is less notable in individuals with severe phenotypes of the disease [64]. Chronic hypoxia induces intensive and continuous erythropoietin production, resulting in the great expansion of the bone marrow (25–30 times), subsequent skeletal deformities and the loss of the bone mass [63, 64, 68]. Simultaneously, a compensatory extramedullary hematopoiesis occurs, creating organomegaly, predominantly of spleen and liver [68]. Nevertheless, if the stimulus is extremely potent, all the cell in the body that express hematopoietic potential will be affected, resulting in the formation of the pseudotumors [78]. Hemolysis will trigger the formation of the gall stones and cholelithiasis and also contributes to splenomegaly development. Besides, the thalassemia-syndrome is recognized as a hypercoagulable state, since erythroid precursors, during the ineffective erythropoiesis, may become prothrombotic. Moreover, in association with platelets and coagulation disruption, the condition may result in serious vascular manifestations

Besides ineffective erythropoiesis and anemia, iron overload also represents very important mechanism in the pathogenesis of the thalassemia, contributing to development of complications. Iron deposition within the reticuloendothelial system in the transfusion- dependent forms of β-thalassemia (major and intermedia) represents associated and secondary mechanism in the pathogenesis of iron overload. However, it is well defined that the most important pathogenetic factor in the hemochromatosis development represents increased iron absorption [80], due

The apoptosis of the erythroid precursors causes subsequent synthesis and secretion of many factors that most likely inhibit hepcidin synthesis in the liver [62]. Coupled with this, it should be underlined that hepcidin functions as a negative iron regulator, delivering the information between the liver and the red blood cells. Its decreased concentrations result in the increased dietary iron absorption and in release of the iron from its storage (macrophages and hepatocytes). The final result is paradoxically and significant dietary iron absorption, regardless of the iron tissue deposition due to the blood transfusions and eventually

The identified molecules that are released from the apoptotic erythroid precursors are growth differentiation factor 15, twisted gastrulation 1, and erythroferrone, and all function as hepcidin expression inhibitors [68, 80–83]. The results have been conflicting so far, since some research demonstrated their significant increase in individuals with β-thalassemia [81, 82], while the others confirmed only increase of erythroferrone in animal models [84]. However, their exact function in the pathogenesis is yet to be elucidated. Nevertheless, the substitution of the synthetic hepcidins represents justified therapy option in patients with the severe forms, as already proven experimentally. However, this extensive and progressive iron overload in synergy with anemia may deteriorate already insufficient hematopoiesis. Iron overload, regardless of its pathogenesis, leads to hemochromatosis and organ

severe forms of β-thalassemias these pathways are inefficient [62].

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

affected nor a carrier [63, 64].

classified as secondary modifiers [62, 68].

of a need for blood transfusion [73].

**4.4 Pathophysiology**

**4.3 Genetic modifiers**

or small oligonucleotides causing the frameshifts in genes that code β-chains. The typical genetic abnormalities that were described are promoter mutations, being responsible for the milder phenotypes, whereas nonsense, initiation codon, splicing and frameshift mutations have been documented in more severe forms of thalassemia-syndromes, characterized with the complete absence of β-chains [62, 63]. Deletions of the gene are randomly identified aberrations, where the deletional removal of one or several genes from the chromosome 11 causes very rare forms of

An autosomal recessive pattern of thalassemia inheritance implicates that both parents have to be heterozygotes, owing a copy of a β-globin gene mutation. Possible outcomes in the affected family may be that every child has: 1) 25% chance of being affected, 2) 50% of being an symptom free and carrier, and 3) 25% of not being

Pathophysiological perception why individuals with beta-thalassemia syndromes may clinically appear very heterogeneous, is based on the perseverance of three group of factors that may modify the disease. These factors are designated as genetic modifiers and are explained as genetic variants that induce differences in disease phenotype [64]. Genetic variants that may impact the imbalance of globin chains are categorized as primary modifiers. The other pathogenetic factors that may alleviate the severity of β-thalassemia major are: coinheritance of an ἀ-thalassemia gene and fetal Hgb production, within the β-globin cluster and are

Coexistence of ἀ-thalassemia enables decreased ἀ-globin chain synthesis, therefore significantly reduces imbalance between the ἀ/non-ἀ-chain in erythrocytes [63]. Increased γ-chain synthesis, in adult life, encounters the excess of ἀ-chains, therefore enables the survival of the erythrocytes that contain fetal hemoglobin, marked as HbF cells. It may be that deletion mutation or point mutation within the β-globin gene cluster simultaneously trigger a rise in fetal Hgb production [62]. According to some research, the increase of HbF synthesis indicates a single nucleotide polymorphism in one of the γ-globin gene promoters or somewhere in the globin locus, resulting in the overexpression of the related gene [62]. It was reported that HbF, that is highly predominant in individuals with severe forms of thalassemia, may account for their improved survival [71]. Moreover, the inverse correlation of HbF levels and factors that reflect disease morbidity was observed, so as the finding that milder phenotypes present with the increased numbers of HbF cells [62, 72]. In addition to this knowledge, it was suggested that certain therapeutic treatments (hydroxyurea) may induce the production of HbF, hence produce less

Tertiary modifiers are recognized to be genetic and environmental factors that modulate disease complication rates. The results of the molecular studies revealed genetic polymorphisms as possible pathogenetic factors involved in cardiac iron overload, hyperbilirubinemia, and Gilbert disease, osteoporosis, and infections susceptibility, that occur in patients with β-thalassemia syndromes [63, 68, 74–76].

Essential pathophysiological determinant in β-thalassemia syndromes is the uncoupling of the synthesis of the ἀ- and the β-chain, where β-chain synthesis is reduced or absent, resulting in the accumulation of ἀ-globin tetramers in the erythroid precursors [62–64]. This phenomenon eventually leads to an ineffective

thalassemias, designated as δβ-, γδβ- and εγδβ-thalassemia [62].

**152**

erythropoiesis, that is a key feature responsible for various pathophysiological consequences during the course of the disease. Erythrocytes and its precursors (mostly polychromatophilic erythroblasts) are filled with precipitated ἀ-globin tetramers, forming inclusive bodies, causing oxidative membrane damage and subsequent apoptosis [62–64, 77]. Physiologically, biochemical detoxification would be efficient to eliminate harmful proteins from the affected cells. Nevertheless, in the severe forms of β-thalassemias these pathways are inefficient [62].

Premature erythroid cell death in the bone marrow (ineffective erythropoiesis) and in the peripheral blood (hemolysis) cause chronic microcytic-hypochromic hemolytic anemia, that is a persistent finding in persons with thalassemia. Interestingly, hemolysis is less notable in individuals with severe phenotypes of the disease [64]. Chronic hypoxia induces intensive and continuous erythropoietin production, resulting in the great expansion of the bone marrow (25–30 times), subsequent skeletal deformities and the loss of the bone mass [63, 64, 68]. Simultaneously, a compensatory extramedullary hematopoiesis occurs, creating organomegaly, predominantly of spleen and liver [68]. Nevertheless, if the stimulus is extremely potent, all the cell in the body that express hematopoietic potential will be affected, resulting in the formation of the pseudotumors [78]. Hemolysis will trigger the formation of the gall stones and cholelithiasis and also contributes to splenomegaly development. Besides, the thalassemia-syndrome is recognized as a hypercoagulable state, since erythroid precursors, during the ineffective erythropoiesis, may become prothrombotic. Moreover, in association with platelets and coagulation disruption, the condition may result in serious vascular manifestations such as venous thrombosis [63, 64, 68, 79].

Besides ineffective erythropoiesis and anemia, iron overload also represents very important mechanism in the pathogenesis of the thalassemia, contributing to development of complications. Iron deposition within the reticuloendothelial system in the transfusion- dependent forms of β-thalassemia (major and intermedia) represents associated and secondary mechanism in the pathogenesis of iron overload. However, it is well defined that the most important pathogenetic factor in the hemochromatosis development represents increased iron absorption [80], due to the hepcidin downregulation and its deficiency [62, 63].

The apoptosis of the erythroid precursors causes subsequent synthesis and secretion of many factors that most likely inhibit hepcidin synthesis in the liver [62]. Coupled with this, it should be underlined that hepcidin functions as a negative iron regulator, delivering the information between the liver and the red blood cells. Its decreased concentrations result in the increased dietary iron absorption and in release of the iron from its storage (macrophages and hepatocytes). The final result is paradoxically and significant dietary iron absorption, regardless of the iron tissue deposition due to the blood transfusions and eventually hemochromatosis [62].

The identified molecules that are released from the apoptotic erythroid precursors are growth differentiation factor 15, twisted gastrulation 1, and erythroferrone, and all function as hepcidin expression inhibitors [68, 80–83]. The results have been conflicting so far, since some research demonstrated their significant increase in individuals with β-thalassemia [81, 82], while the others confirmed only increase of erythroferrone in animal models [84]. However, their exact function in the pathogenesis is yet to be elucidated. Nevertheless, the substitution of the synthetic hepcidins represents justified therapy option in patients with the severe forms, as already proven experimentally. However, this extensive and progressive iron overload in synergy with anemia may deteriorate already insufficient hematopoiesis. Iron overload, regardless of its pathogenesis, leads to hemochromatosis and organ damage [83, 85].

#### **4.5 Clinical findings**

The main clinical features of β-thalassemia syndromes are anemia and iron overload, leading to severe and life threating consequences. The onset and the degree of the symptoms severity depend whether the affected individuals present as a homozygous phenotype (thalassemia major) or as a homozygotes or compound heterozygotes (thalassemia intermedia). Correspondingly, individuals with β-thalassemia minor are usually asymptomatic and may be discovered incidentally, having only the discrete changes in the hematological findings.

The onset of symptoms will appear 12 months after the birth, [67–85], at the moment when HbF production switches to adult and physiological synthesis of HbA is yet to be established [86]. The infants will experience feeding problems, recurrent fevers, diarrhea, enlargement of the abdomen and the growth retardation. If the child has not been diagnosed prenatally, this is the point when the diagnosis of thalassemia is determined, and transfusion indicated [63, 64].

Microcytic-hypochromic hemolytic anemia is an obligatory finding in the affected individuals, predisposing them to progressive paleness and jaundice. Bone marrow expansion secondary to erythroid hyperplasia, lead to significant skeletal changes, creating abnormalities of the face and body. People with severe phenotypes most often experience frontal bossing, depression of the bridge of the nose, mandible and maxilla enlargement with the upper teeth exposure, bone pain, osteopenia and osteochondrosis. If spinal impairment occurs during the childhood, linear growth is delayed, resulting in the discordance in the length of upper and lower limbs [63, 64, 87]. The progressive enlargement of the abdomen is due to the hepatosplenomegaly, whereas the masses of extramedullar hematopoietic tissue may also be found in the chest or spinal column [63, 64].

Iron overload predominates in the most severe clinical phenotypes. Brown pigmentation of the skin, particularly in the areas exposed to the sun, reflects systemic hemochromatosis. Predominant sites for iron deposition tend to be spleen, liver, myocardium, pancreas, and endocrine glands. Although significant liver deposition of iron could be found, its function may be preserved for a long time [62]. Ultimately, liver cirrhosis may develop. Cardiac manifestations stand for the most adverse outcome of iron overload, whereas arrhythmias, dilated cardiomyopathy, and atrial or/and ventricular failure during the course of the disease lead to congestive cardiac failure. Endocrine complications primarily develop due to the insufficiency of the growth hormone (growth retardation) and sex hormones (hypogonadism). Additionally, hormone substitution therapy is commonly required for maintaining normal fertility. Other endocrine disturbances may be very diverse, including diabetes mellitus, hypothyroidism, hypoparathyroidism, hypocorticism, and panhypopituitarism. Pulmonary hypertension may contribute to the complexity of the cardiovascular manifestations by deteriorating left heart function [79, 88].

Other clinical features in β-thalassemia syndromes are osteoporosis, subclinical fractures, nutritional deficiencies, venous thrombosis, chronic B and/or C hepatitis, and infections. The risk of hepatocellular carcinoma in patients who develop liver cirrhosis remains unchanged even if the proper therapy is performed, due to the oxidative DNA damage triggered by chronic iron accumulation [79, 88].

#### **4.6 Laboratory findings and Hgb analysis**

Laboratory diagnosis of thalassemia is confirmed based on established red blood cells parameters, qualitative and quantitative Hgb analysis and, when necessary, molecular assessment. Erythrocyte count may be relatively high, whereas Hgb is

**155**

cause of mortality [63].

high-income countries [92].

*Adaptation to Mediterranea*

**4.7 Therapy approach**

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

reduced <7 mg/dL, mean corpuscular volume (MCV) is between 50 and 70 fL and mean corpuscular Hgb (MCH) 12-20 pg. Peripheral blood smear demonstrates microcytosis, hypochromia, anisocytosis, poikilocytosis (dacrocytes and elliptocytes), along with the erythroblasts. The number of reticulocytes may remain normal, without any diagnostic accuracy. In order to differentiate iron deficiency anemia form the thalassemia-syndromes, few formulas are available to calculate a thalassemic index, but should be performed with caution [63, 64, 89]. In biochemical terms, typical β-thalassemia presents with elevated ferritin levels >12 ng/mL, transferrin saturation increased to 75-100% and unconjugated hyperbilirubinemia [62, 88].

The most accurate method for β-thalassemia differentiation is quantitative HbA2 determination. Considering that physiological HbF in adult population is commonly less than 1.5%, the results for HbA2 ranging between 3.6 and 7% are considered as definite thalassemia values. Nonconclusive or borderline cases, with HbA2 ranging between 3.2 and 3.6%, respectively, require further analysis [86, 89]. Additionally, PCR-based procedures or β-globin gene sequence analysis are necessary for diagnosis confirmation. Besides, in couples with increased risk, a prenatal diagnosis of thalassemia may be achieved by chorionic villi sampling (11th gestational week) or DNA analysis from harvested fetal cells (15-18th gestational week) [63, 64, 89].

Conventional management of β-thalassemia syndromes includes blood transfusion, iron chelation, splenectomy and hemopoietic stem-cell transplantation. The introduction of blood transfusion in regular management of β-thalassemia has enormously improved quality of life and survival of the affected individuals [62–68]. The mayor indication for its initiation, in previously diagnosed patients, should be low Hgb level (<7 g/dL), that lasts at least two weeks [64], concerning other clinical signs such as growth retardation, skeletal changes and splenomegaly. The therapeutic aim of transfusion is to maintain Hgb level at 9-10 g/dL or 11-12 g/ dL in cases of confirmed cardiovascular disease [63, 64, 68, 86]. Although lifesaving approach, blood transfusion has several adverse effects, with iron overload

and viral infections (hepatitis B, C) being the most common [62–68].

The knowledge that iron cannot be excreted form the human body and that patients requiring constant blood transfusions tend to develop iron overload, lead to the regular assessment of iron body status. Most conventional method is determination of serum ferritin levels, that may be monitored in order to initiate chelation therapy or may be used as a biomarker of iron chelators efficiency. However, more reliable, yet non-invasive method of tissue iron accumulation has been developed. Magnetic resonance imaging has been successfully used for liver and cardiac iron overload, measuring a tissue iron concentration in mg of iron per gr of dry liver/ heart weight [63, 90, 91]. Also, iron binders (chelators) enable its elimination through feces and/or urine and should be initiated after approximately 10-20 performed transfusions or with ferritin levels above 1000 mg/gL [64, 68].

Splenectomy is indicated in the following cases: enlarged spleen with the risk of rupture, severe cytopenia and in patients with the significant blood requirements. In patients with splenectomy, infections and subsequent sepsis remain the leading

However, the only curable therapy for the thalassemia represents hematopoietic

stem-cell transplantation [63, 64, 68]. Nevertheless, it was documented that a disease-free survival may be achieved in 80% in matched donors and even 65% in unrelated donors and umbilical blood cord stem-cells transplantation. Nevertheless, this therapy option is still associated with risk and complications, even in the

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

**4.5 Clinical findings**

The main clinical features of β-thalassemia syndromes are anemia and iron overload, leading to severe and life threating consequences. The onset and the degree of the symptoms severity depend whether the affected individuals present as a homozygous phenotype (thalassemia major) or as a homozygotes or compound heterozygotes (thalassemia intermedia). Correspondingly, individuals with β-thalassemia minor are usually asymptomatic and may be discovered incidentally,

The onset of symptoms will appear 12 months after the birth, [67–85], at the moment when HbF production switches to adult and physiological synthesis of HbA is yet to be established [86]. The infants will experience feeding problems, recurrent fevers, diarrhea, enlargement of the abdomen and the growth retardation. If the child has not been diagnosed prenatally, this is the point when the diagnosis of thalassemia is determined, and transfusion indicated [63, 64]. Microcytic-hypochromic hemolytic anemia is an obligatory finding in the affected individuals, predisposing them to progressive paleness and jaundice. Bone marrow expansion secondary to erythroid hyperplasia, lead to significant skeletal changes, creating abnormalities of the face and body. People with severe phenotypes most often experience frontal bossing, depression of the bridge of the nose, mandible and maxilla enlargement with the upper teeth exposure, bone pain, osteopenia and osteochondrosis. If spinal impairment occurs during the childhood, linear growth is delayed, resulting in the discordance in the length of upper and lower limbs [63, 64, 87]. The progressive enlargement of the abdomen is due to the hepatosplenomegaly, whereas the masses of extramedullar hematopoietic tissue

Iron overload predominates in the most severe clinical phenotypes. Brown pigmentation of the skin, particularly in the areas exposed to the sun, reflects systemic hemochromatosis. Predominant sites for iron deposition tend to be spleen, liver, myocardium, pancreas, and endocrine glands. Although significant liver deposition of iron could be found, its function may be preserved for a long time [62]. Ultimately, liver cirrhosis may develop. Cardiac manifestations stand for the most adverse outcome of iron overload, whereas arrhythmias, dilated cardiomyopathy, and atrial or/and ventricular failure during the course of the disease lead to congestive cardiac failure. Endocrine complications primarily develop due to the insufficiency of the growth hormone (growth retardation) and sex hormones (hypogonadism). Additionally, hormone substitution therapy is commonly required for maintaining normal fertility. Other endocrine disturbances may be very diverse, including diabetes mellitus, hypothyroidism, hypoparathyroidism, hypocorticism, and panhypopituitarism. Pulmonary hypertension may contribute to the complexity of the cardiovascular manifestations by deteriorating left heart

Other clinical features in β-thalassemia syndromes are osteoporosis, subclinical fractures, nutritional deficiencies, venous thrombosis, chronic B and/or C hepatitis, and infections. The risk of hepatocellular carcinoma in patients who develop liver cirrhosis remains unchanged even if the proper therapy is performed, due to the

Laboratory diagnosis of thalassemia is confirmed based on established red blood cells parameters, qualitative and quantitative Hgb analysis and, when necessary, molecular assessment. Erythrocyte count may be relatively high, whereas Hgb is

oxidative DNA damage triggered by chronic iron accumulation [79, 88].

**4.6 Laboratory findings and Hgb analysis**

having only the discrete changes in the hematological findings.

may also be found in the chest or spinal column [63, 64].

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function [79, 88].

reduced <7 mg/dL, mean corpuscular volume (MCV) is between 50 and 70 fL and mean corpuscular Hgb (MCH) 12-20 pg. Peripheral blood smear demonstrates microcytosis, hypochromia, anisocytosis, poikilocytosis (dacrocytes and elliptocytes), along with the erythroblasts. The number of reticulocytes may remain normal, without any diagnostic accuracy. In order to differentiate iron deficiency anemia form the thalassemia-syndromes, few formulas are available to calculate a thalassemic index, but should be performed with caution [63, 64, 89]. In biochemical terms, typical β-thalassemia presents with elevated ferritin levels >12 ng/mL, transferrin saturation increased to 75-100% and unconjugated hyperbilirubinemia [62, 88].

The most accurate method for β-thalassemia differentiation is quantitative HbA2 determination. Considering that physiological HbF in adult population is commonly less than 1.5%, the results for HbA2 ranging between 3.6 and 7% are considered as definite thalassemia values. Nonconclusive or borderline cases, with HbA2 ranging between 3.2 and 3.6%, respectively, require further analysis [86, 89]. Additionally, PCR-based procedures or β-globin gene sequence analysis are necessary for diagnosis confirmation. Besides, in couples with increased risk, a prenatal diagnosis of thalassemia may be achieved by chorionic villi sampling (11th gestational week) or DNA analysis from harvested fetal cells (15-18th gestational week) [63, 64, 89].

### **4.7 Therapy approach**

Conventional management of β-thalassemia syndromes includes blood transfusion, iron chelation, splenectomy and hemopoietic stem-cell transplantation. The introduction of blood transfusion in regular management of β-thalassemia has enormously improved quality of life and survival of the affected individuals [62–68]. The mayor indication for its initiation, in previously diagnosed patients, should be low Hgb level (<7 g/dL), that lasts at least two weeks [64], concerning other clinical signs such as growth retardation, skeletal changes and splenomegaly. The therapeutic aim of transfusion is to maintain Hgb level at 9-10 g/dL or 11-12 g/ dL in cases of confirmed cardiovascular disease [63, 64, 68, 86]. Although lifesaving approach, blood transfusion has several adverse effects, with iron overload and viral infections (hepatitis B, C) being the most common [62–68].

The knowledge that iron cannot be excreted form the human body and that patients requiring constant blood transfusions tend to develop iron overload, lead to the regular assessment of iron body status. Most conventional method is determination of serum ferritin levels, that may be monitored in order to initiate chelation therapy or may be used as a biomarker of iron chelators efficiency. However, more reliable, yet non-invasive method of tissue iron accumulation has been developed. Magnetic resonance imaging has been successfully used for liver and cardiac iron overload, measuring a tissue iron concentration in mg of iron per gr of dry liver/ heart weight [63, 90, 91]. Also, iron binders (chelators) enable its elimination through feces and/or urine and should be initiated after approximately 10-20 performed transfusions or with ferritin levels above 1000 mg/gL [64, 68].

Splenectomy is indicated in the following cases: enlarged spleen with the risk of rupture, severe cytopenia and in patients with the significant blood requirements. In patients with splenectomy, infections and subsequent sepsis remain the leading cause of mortality [63].

However, the only curable therapy for the thalassemia represents hematopoietic stem-cell transplantation [63, 64, 68]. Nevertheless, it was documented that a disease-free survival may be achieved in 80% in matched donors and even 65% in unrelated donors and umbilical blood cord stem-cells transplantation. Nevertheless, this therapy option is still associated with risk and complications, even in the high-income countries [92].

Considering the monogenic nature of the disease, the most challenging, yet possible therapy approach, may be an interference in the globin chains imbalance, achieved by gene therapy and genome editing [68]. Alternative pharmaceutical approaches would be use of agents acting as potent stimulators of late stage erythropoiesis and increased hepcidin expression, throughout its substitution or stimulation of its endogenous production. Even though there has been a substantial progress in the development of therapy options for individuals affected with thalassemia, the best approach to the disease management remains prevention of thalassemia births throughout national screening programs [68].
