Laboratory Diagnosis of β-Thalassemia and HbE

*Thanusak Tatu*

#### **Abstract**

β-Thalassemia and HbE, each, is a syndrome resulted from quantitative and qualitative defects of β-globin chain, respectively. In addition to history retrieve and physical examination, diagnosis of these disorders requires laboratory information. Laboratory tests that are conventionally performed to diagnose the β-thalassemia and HbE are classified into two groups, based on the purposes, including the screening tests and confirmatory tests. The screening tests are aimed to screen for carriers of the β-thalassemia and HbE, while confirmatory tests are the tests performed to definitely diagnose these disorders. This chapter will explain all of these tests, the information of which will be useful for those who are working and interested in the β-thalassemia and HbE.

**Keywords:** β-thalassemia, HbE, screening tests, confirmatory tests, thalassemia carrier, HbE carrier, β-thalassemia disease

#### **1. Introduction**

#### **1.1 Thalassemia and hemoglobinopathies**

Thalassemia is a type of anemia caused by reduction or absence of globin chain synthesis, which results in imbalanced-globin chain synthesis; the major pathogenesis of the disease. The unaffected globin chains continuing to be synthesized at the normal synthetic rate tend to form homotetramers and aggregation that can harm the red blood cells both at young and mature stages. The α-globin chain aggregation formed in β-thalassemia causes ineffective erythropoiesis due to oxidative stress. The γ- and β-globin chain homotetramer (γ4, β4) formed in α-thalassemia harm mature erythrocytes. The γ4 or Hb Bart's has very high oxygen affinity and inhibits oxygen release from erythrocytes which, in turn, results in tissue anoxia. The β4 or HbH is an unstable hemoglobin and precipitates easily under the stress condition in the body. Once precipitates, the erythrocytes are removed by the RE system resulting in anemia.

Severe thalassemia cases suffer from chronic and marked anemia with life relying solely on blood transfusion. Anemia causes expansion of bone marrow, leading to osteoporosis and changes of bone structure. Blood transfusion and hemolysis cause iron overloading state in the body which causes several complications such as heart disease, growth retardation, diabetes mellitus, and infection.

Thalassemia is considered the most common autosomal single-gene disorder worldwide. It can be found in more than 150 countries with an estimated carrier frequency of about 7%. The Mediterranean region, certain parts of North and West Africa, Middle East, Indian subcontinent, Southern Far East, and South East Asia have the highest prevalence of the disease [1].

In contrast to the thalassemia, hemoglobinopathy is an inherited disorder of hemoglobin productions characterized by production of abnormal hemoglobin or hemoglobin structural variants occurring from genetic alterations including point mutations, deletions or insertion of the normal globin genes. The well-known abnormal hemoglobins in the world are HbS, which is common in western countries as well as in the Middle East and HbE, which is common in Southeast Asia [2].

#### **1.2 Types of thalassemia and hemoglobinopathies**

Two major types of thalassemia are found across the world consisting of the α- and β-thalassemia. The α-thalassemia is further sub-divided into two types: α-thalassemia 1 or αO-thalassemia and α-thalassemia 2 or α<sup>+</sup> -thalassemia. Gene deletion is the leading cause of α-thalassemia. In α-thalassemia 1, two α-globin gene in-cis on chromosome 16 are deleted, while only one α-globin gene is absent in the α-thalassemia 2 [3]. In addition, there are two types of β-thalassemia; βO-thalassemia and β<sup>+</sup> -thalassemia. In contrast to α-thalassemia, mutations on the β-globin gene are found in the majority of the β-thalassemia patients [4].

Two broad types of hemoglobinopathies or structural variants are also found; α- and β-hemoglobinopathies. To date, approximately 1358 structural variants are described (http://globin.bx.psu.edu), around 90% of which are involved in the β, γ, and δ-globin chains and around 60% involves the α-globin chain. The most important β-globin hemoglobinopathies are HbS (α2β<sup>2</sup> 6Glu−Val**)** and HbE (α<sup>2</sup> β2 26Glu−Lys). Hb E is found around the world and accounts for approximately 13–17% on the population of Thailand, especially in the Thai-Laos-Cambodian boundary or "Hb E triangle" where more than 32–60% of the people carry HbE gene [5].

#### *1.2.1 β-Thalassemia*

The β-thalassemia is a diverse group of disorders of hemoglobin synthesis which is characterized by reduced or absent β-globin chain synthesis. There are two main types of β-thalassemia: βO-thalassemia in which no β-globin chain is produced and β+ -thalassemia in which some β-globin is produced but less than normal.

β0 -Thalassemia is severe β-thalassemia with no production of β-globin chain. It is mainly caused by point mutations in coding region (exon) or exon-intron junction of β-globin gene which lead to premature stop codon or generation of abnormal β-globin mRNA. The end results of these abnormalities are absence of the β-globin chain production [6]. In Thailand, at least three common mutations in the β-globin gene are of this category. They comprise A-T substitution at codon 17 (CD17: A-T) which creates premature stop codon, the TTCT-deletion at codons 41/42 (CD41/42: −TTCT) which causes reading frameshift and premature stop codon at codon 59 instead of codon 147, the G-T substitution at IVSI-nt1 which leads to abnormal splicing of immature β-mRNA and results in no production of normal β-mRNA. In general, thus, genotype of heterozygote is written as β<sup>0</sup> /βA and that for homozygote as β<sup>0</sup> /β0 [2, 7–9].

β+ -Thalassemia is a milder form of β-thalassemia in which some β-globin chains are still produced. The majority of cases possess point mutations outside exons, especially in the promoter region. The mutations of β-globin gene leading to the β+ -thalassemia include mutations at ATA box (nt-28, nt-29 or nt-30 from cap site), CACCC element (nt-86 to nt-90 from cap site), and mutations in introns or exons of gene to produce new splice site to race in RNA splicing process, as mutation in IVS2-nt654 (C-T) and mutation of IVS1-nt5 (G-C) [2, 6]. The genotypes were β<sup>+</sup> /β<sup>A</sup> and β<sup>+</sup> /β+ for heterozygote and homozygote consecutively.

**125**

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

across the world (http://globin.cse.psu.edu/).

mately 8–70% of population [11].

**1.4 Inheritance of genes for β-thalassemia and HbE**

β-thalassemia trait. Those who are heterozygote for HbE gene (β<sup>E</sup>

β-Hemoglobinopathies are characterized by the production of abnormal β-globin chains due to changes or mutations (missense mutations) on the β-globin gene. Two abnormal β-globin chains then assemble with two normal α-globin chains to form abnormal hemoglobin or β-structural variants. These abnormal hemoglobins generally have different electrophysical properties from their normal counterparts; that is, due to the molecular conformational alteration. In theory, synthetic rate of the abnormal β-globin chain should be normal. However, some are produced in reduced rate, thus producing a phenotype resembling the β<sup>+</sup>

thalassemia. To date, there are more than 737 β-globin structural variants reports

HbE is abnormal hemoglobin resulted from the G-A substitution at codon 26 of β-globin gene. This missense mutation partially activates a cryptic splice site toward the 3′ end of exon 1, resulting in a proportion of abnormally splice mRNA. Thus,

 globin is synthesized, leading to a mild thalassemia phenotype. HbE is becoming the common β-globin structure variant across the world as a result of migration and inter racial marriage [2, 10]. It has been realized to be the hallmark of Southeast Asian region. In Thailand, HbE is very common accounting for approxi-

Gene for β-thalassemia and HbE is transmitted within the family from parents to descendants in an autosomal recessive fashion. Thus, those who are heterozygous for abnormal β-gene are clinically asymptomatic and called β-thalassemia carrier or

cally asymptomatic and called HbE carrier or HbE trait. However, homozygote or compound heterozygote of the β-thalassemia gene and/or HbE gene are clinically affected and suffer from chronic anemia with some life-threatening complication. Therefore, accurate diagnosis of carriers of the β-thalassemia and HbE as well as the

The carriers of β-thalassemia and HbE do not have clinical burden as they are clinically normal and have normal quality of life. However, if the β-thalassemia carriers get married with the HbE carriers, they will have 25% chance of producing the HbE/β-thalassemia babies. The HbE/β-thalassemia or sometimes called the β-thalassemia/HbE disease is a thalassemia syndrome that presently is known to be clinically heterogeneous [10–12]. Some patients are very mild, while some are very severe. The severe cases always required regular blood transfusion which always ends up with iron overloading condition. Without proper management of this iron overloading, several fatal complications occur, leading to low quality of life and,

The diagnosis of β-thalassemia and HbE involves both clinical and laboratory investigations. Clinical data can only identify the affected patients, but cannot


) are also clini-

*1.2.2 β-Hemoglobinopathies*

**1.3 Hemoglobin E**

disease is important.

**1.5 Problem of β-thalassemia and HbE**

finally, pre-death at young age.

**1.6 Diagnosis of β-thalassemia and HbE**

less β<sup>E</sup>

### *1.2.2 β-Hemoglobinopathies*

*Beta Thalassemia*

patients [4].

*1.2.1 β-Thalassemia*

β+

β0

In contrast to the thalassemia, hemoglobinopathy is an inherited disorder of hemoglobin productions characterized by production of abnormal hemoglobin or hemoglobin structural variants occurring from genetic alterations including point mutations, deletions or insertion of the normal globin genes. The well-known abnormal hemoglobins in the world are HbS, which is common in western countries as well as in the Middle East and HbE, which is common in Southeast Asia [2].

Two major types of thalassemia are found across the world consisting of the α- and β-thalassemia. The α-thalassemia is further sub-divided into two

Gene deletion is the leading cause of α-thalassemia. In α-thalassemia 1, two α-globin gene in-cis on chromosome 16 are deleted, while only one α-globin gene is absent in the α-thalassemia 2 [3]. In addition, there are two types of

mutations on the β-globin gene are found in the majority of the β-thalassemia

Hb E is found around the world and accounts for approximately 13–17% on the population of Thailand, especially in the Thai-Laos-Cambodian boundary or "Hb E

The β-thalassemia is a diverse group of disorders of hemoglobin synthesis which is characterized by reduced or absent β-globin chain synthesis. There are two main types of β-thalassemia: βO-thalassemia in which no β-globin chain is produced and




are still produced. The majority of cases possess point mutations outside exons, especially in the promoter region. The mutations of β-globin gene leading to the

for heterozygote and homozygote consecutively.

triangle" where more than 32–60% of the people carry HbE gene [5].


Two broad types of hemoglobinopathies or structural variants are also found; α- and β-hemoglobinopathies. To date, approximately 1358 structural variants are described (http://globin.bx.psu.edu), around 90% of which are involved in the β, γ, and δ-globin chains and around 60% involves the α-globin chain. The most impor-



6Glu−Val**)** and HbE (α<sup>2</sup>

β2

/βA and that for homozygote

/β<sup>A</sup>

26Glu−Lys).

types: α-thalassemia 1 or αO-thalassemia and α-thalassemia 2 or α<sup>+</sup>

**1.2 Types of thalassemia and hemoglobinopathies**

β-thalassemia; βO-thalassemia and β<sup>+</sup>

tant β-globin hemoglobinopathies are HbS (α2β<sup>2</sup>

general, thus, genotype of heterozygote is written as β<sup>0</sup>

**124**

and β<sup>+</sup> /β+

as β<sup>0</sup> /β0

β+

β+

[2, 7–9].

β-Hemoglobinopathies are characterized by the production of abnormal β-globin chains due to changes or mutations (missense mutations) on the β-globin gene. Two abnormal β-globin chains then assemble with two normal α-globin chains to form abnormal hemoglobin or β-structural variants. These abnormal hemoglobins generally have different electrophysical properties from their normal counterparts; that is, due to the molecular conformational alteration. In theory, synthetic rate of the abnormal β-globin chain should be normal. However, some are produced in reduced rate, thus producing a phenotype resembling the β<sup>+</sup> thalassemia. To date, there are more than 737 β-globin structural variants reports across the world (http://globin.cse.psu.edu/).
