Beta-Thalassemia

Beta thalassemia

What is beta thalassemia? Beta thalassemia, a form of heterogeneous hereditary anemia, arises from the deficiency or absence of β-globin chain production in individuals. The normal state of hemoglobin consists of two α chains and two β chains, with the α chain synthesis genes located on chromosome 16 and the β chain genes situated on chromosome 11. Mutation in the HBB gene on chromosome 11 leads to gene dysfunction and the manifestation of various types of beta thalassemia. Hemoglobin protein plays a crucial role in binding and transporting oxygen to different body tissues. In individuals affected by beta thalassemia, the insufficiency or absence of beta globin hampers the normal growth of red blood cells, resulting in diverse symptoms, including anemia.

Approximately 68,000 children worldwide are born with different forms of thalassemia each year, making it one of the most prevalent diseases globally. Beta thalassemia follows an autosomal recessive inheritance pattern, with an estimated 80 to 90 million people (1.5% of the global population) being carriers of the condition. The heightened frequency of hemoglobin-related diseases compared to other monogenic disorders underscores the role of natural selection and adaptation of carriers against malaria. Historically, beta thalassemia has exhibited a remarkably high prevalence in various regions such as the Mediterranean, the Middle East and Southeast Asia, which can be attributed to gene drift and founder effects.

Types of beta thalassemia

The occurrence of beta thalassemia disease arises from the diminution or absence of β chain production in individuals. Consequently, there are distinct types of this disease based on its severity and the nature of the mutations. These types consist of the following:

1) Beta thalassemia major

The most severe variant of beta thalassemia is its major type, also referred to as Cooley’s anemia. This particular condition manifests when both beta globin genes undergo mutation, resulting in the absence of protein production. Typically, symptoms emerge in individuals afflicted with this disease between the ages of 6 months and 2 years, necessitating continuous blood transfusions.

The level of hemoglobin may drop below 7 g/dL and Hb F may be less than 90%. As a means of compensating for the depletion of red blood cells, the bone marrow expands, thereby giving rise to symptoms such as bone abnormalities, spleen enlargement and restricted growth. Due to the regular blood transfusions required by individuals with beta thalassemia major, various organs may exhibit iron overload, leading to the development of disorders such as diabetes, liver cirrhosis, heart arrhythmia, dark skin pigmentation, hypothyroidism, hypophysis and myopathy.

Symptoms:

Abdominal swelling
Frequent infections
Decreased growth rate
Loss of appetite
Pale skin
Yellow skin color
Thinning of bones
Muscle weakness
Hepatosplenomegaly (enlargement of the liver and spleen)
Occurrence of skeletal and cranial changes such as deformity of long bones and forehead protrusion
wounds in the leg
Osteoporosis

Alleles:

β^o/β^o
β⁺/β⁺

2) β-thalassemia intermedia

This particular form of beta thalassemia, referred to as TI, presents with a less severe form of anemia, thus regular blood transfusions unnecessary or only sporadically required. Although patients with beta thalassemia may experience an increased iron load in their bloodstream, the presence of hypogonadism and hypothyroidism is not commonly observed. In certain instances, individuals with the intermediate type may remain asymptomatic until adulthood; however, in general, the symptoms of this type of beta thalassemia are very close to thalassemia major. The age at which this disease manifests typically falls between 2 and 6 years.

Symptoms:

Jaundice
Enlargement of the liver and spleen
kidney stone
Moderate to severe skeletal problems
Gallstone
Frequent wounds in the leg
Pale skin

Alleles:

β⁺/β⁺
β⁰/β⁺

3) beta thalassemia minor or beta thalassemia trait

Carrier beta thalassemia or trait beta thalassemia are alternative names for this particular form of beta thalassemia. It occurs when only one copy of the beta globin gene is mutated, while the other copy remains unaltered. This condition often manifests without any clinical symptoms and is typically triggered by physiological stress, pregnancy or during childhood. Beta thalassemia minor usually does not exhibit any specific symptoms, with only a slight presence of blood, caused by an abnormality in the appearance or morphology of erythrocytes, being observed. The hemoglobin level in these cases often exceeds 10 g/dl. If both parents are carriers of beta thalassemia, there is a 25% chance that their child will be homozygous for beta thalassemia.

Alleles:

β/β⁺
β/β⁰

The cause and inheritance pattern in beta thalassemia

The beta globin coding gene is located on the short arm of chromosome 11, alongside other genes such as the δ-globin gene, the embryonic ɛ gene, the embryonic A-γ and G-γ genes, as well as a pseudogene (ψB1). These genes are located in the designated region. The HBB gene, which spans approximately 1.6 kb, encompasses 3 exons, 5′-UTR and 3′-UTR. The expression of this gene is regulated by a promoter region containing a TATA Box, CAAT Box and CACCC Box. The primary regulatory sequence, known as the locus control region, is located 50 kb away from the HBB gene. This region consists of 4 erythroid-specific DNAse hypersensitive sites (HS-1 to HS-4), which serve as indicators of DNA-protein interaction.

Each of these loci contains a combination of DNA motifs that interact with transcription factors, including GATA-1, nuclear factor erythroid 2 and KLF1. These transcription factors play a crucial role in binding and regulating gene expression. For instance, the KLF1 transcription factor is involved in the regulation of HBB gene expression by binding to the CACCC Box. In mice, if the KLF1 gene is deactivated or knocked out, clinical manifestations resembling beta thalassemia are observed.

Beta thalassemia is a genetically heterogeneous disorder with an autosomal recessive inheritance pattern. To date, over 200 types of mutations associated with this disease have been documented, and the complete list can be accessed on the Globin Gene Server. Most of the reported mutations in the genes associated with beta thalassemia involve single-nucleotide substitutions, insertions of single nucleotides or small nucleotide insertions that result in frameshift mutations within critical regions of the HBB gene.

the β⁰ state in beta thalassemia disease refers to the complete absence of beta globin chain production, which can arise from various causes such as deletion mutations, frameshift mutation, nonsense, splice-site junction and others. Conversely, the β⁺ state signifies low production and inadequate levels of β-globin chain due to mutations in the promoter region (CACCC Box or TATA Box), polyadenylation signal, splicing abnormalities or mutations in the 3ʹ UTR or 5ʹ-UTR.

Consequently, based on the type of mutations and their impact on β-globin protein production, this disease is classified into different types. The phenotype or clinical manifestations of homozygous beta thalassemia can be influenced by the presence of another genetic abnormality outside the HBB gene cluster. One such gene that can modify the phenotype of beta thalassemia disease is the UGT1A gene, which encodes Uridine diphosphoglucuronyl transferase. When an individual is affected by beta thalassemia major or intermediate and also has a defect in the UGT1A gene, it results in the development of Gilbert disease. The table below outlines several common mutations in the HBB gene and the severity of the disease in countries with a high prevalence of beta thalassemia.

Population	Severity	Mutations in β gene
Mediterranean	β++	−101 C→T
Japan	β++	−31 A→G
India	β0	−619 del
china	β+	IVS2‐nt654 C→T
Africa	β++	−29 A→G
Southeast Asia	β++	−28 A→C
Africa-America	β++	AATAAA to AACAAA

Mutations associated with beta thalassemia

Almost the majority of mutations associated with the disease known as beta thalassemia act by means of down regulation in the cis-acting form and occur in the alleles of the beta globin gene. However, there are some mutations that act in the trans-acting form and affect the expression of this gene. Until now, approximately 300 beta thalassemia alleles have been identified and their complete information can be found on the website (http://globin.cse.psu.edu). In contrast to alpha thalassemia, where the cause of the disease is deletion mutations in the gene cluster of the α-globin gene, in beta thalassemia, most of the mutations are point mutations and mutations in the regions immediately surrounding the β-globin gene, which play a significant role. In general, mutations associated with beta thalassemia can be classified into two groups: deletion and non-deletion types. These groups are further described below.

1) Non-deletion mutations

Non-deletion mutations are the primary types of changes observed in the β-globin gene. These mutations include single nucleotide substitutions, small insertions, and deletions in the gene region or the regions immediately surrounding it. These mutations can affect various steps such as transcription, RNA processing, and mRNA translation of the beta globin gene, resulting in reduced gene expression. These mutations will be discussed in more detail.

Transcriptional Mutants

In this case, point mutations occur in the conserved DNA sequence in the promoter region of the β-globin gene. These mutations are found approximately 100 nucleotides above the transcription start site, where important sequences such as CCAAT, CACCC, and ATAA are located and extend to about 50 nucleotides in the 5′ UTR region. Mutations in the CCAAT box region often lead to milder forms of beta thalassemia. Some beta thalassemia alleles cause very mild symptoms in individuals, to the extent that in heterozygous carriers, the level of HbA2 is normal and they have nearly normal red blood cells. This condition is referred to as “Silent” beta thalassemia and is typically uncommon, except for the -101 C → T mutation, which is prevalent in Mediterranean regions. Other silent mutations include those occurring in the 5′ UTR region, such as CAP +1 (A→C) (common in the Indian population) and CAP +8 (C→T) (common in the Chinese population).

Mutations affecting RNA processing

A wide range of mutations interfere with the processing stage of primary mRNAs. These mutations occur in the region of GT or AG dinucleotides at the exon-intron splice junction, resulting in a complete loss of splicing and leading to the β⁰-thalassemia phenotype. Mutations in this region can be substitutions or small deletions. One example of such mutations is the 5 IVS1 G → C, T, or A, which reduces the efficiency of splicing. The IVS1-110 G to A mutation is the first known base substitution mutation in beta thalassemia and is highly prevalent in the Mediterranean population.

Mutations causing Abnormal Post transcriptional Modification

The mRNA molecule that has recently been synthesized necessitates modification on both its 5′ and 3′ ends for proper functionality. This modification encompasses the addition of a 5′ Cap and a 3′ poly-A tail. The inclusion of the poly-A tail sequence is guided by an AATAAA hexanucleotide sequence positioned approximately 20 nucleotides upstream from the poly-A tail. In the event of a mutation occurring in this specific region, the efficacy of mRNA modification will be diminished, consequently resulting in β⁺-thalassemia.

Mutations affecting β-Globin mRNA translation

Mutations that impact the start codon (ATG) will uniformly give rise to β⁰-thalassemia. Among this group of mutations, one that commonly occurs involves the insertion of a 45 bp sequence between positions -22 and +23, thereby affecting the start codon. A significant proportion of beta thalassemia alleles contain premature termination codons. These codons can originate either from direct mutations leading to the formation of the termination codon or from alterations in the reading frame. One of the extensively studied mutations is observed in codon 39 (CAG to TAG). This mutation represents the second most prevalent beta thalassemia mutation in the Mediterranean region, and it stands as the most frequently encountered mutation in Sardinia.

2) deletion mutations

The presence of deletion mutations in beta thalassemia is a rare occurrence, unless the deletion affects the β-globin gene itself or the mutations impact the upstream βLCR region.

One particular deletion of 150 bp to approximately 67 kb affects the β-globin gene, resulting in the development of β⁰-thalassemia. Another set of deletion mutation occur in the upstream region of the β-globin gene known as βLCR, leading to a reduction in the expression of this gene and other related genes within the gene cluster located on chromosome 11. This specific mutation type gives rise to (εγδβ)0-Thalassemia.

Beta thalassemia prevalence rate

Beta thalassemia is one of the most prevalent diseases in various regions such as the Middle East, India, Central Asia, South China, South America and African coastal countries. These regions, according to the documented statistics, exhibit the highest carrier rates in the Maldives (18%), Cyprus (14%), Sardinia (10.3%) and Southeast Asia. The increased incidence of beta thalassemia and its carriers in these areas can be attributed to the presence of malaria (Plasmodium falciparum) and consanguineous marriages. Annually, approximately 60,000 individuals are born displaying symptoms of this disease, while an estimated 80 to 90 million people worldwide carry the beta thalassemia trait. Generally, the prevalence rate of this disease is 1 case per 10,000 births in Europe and 1 case per 100,000 individuals worldwide. Given the autosomal nature of this condition, it is unaffected by gender, impacting both males and females alike.

Other names of beta thalassemia

Erythroblastic anemia
Mediterranean anemia
Thalassemia, beta type

Beta thalassemia diagnosis methods

There exist numerous laboratory and genetic techniques for the identification of beta thalassemia. Standard laboratory examinations employed in the diagnosis of this condition encompass complete blood count (CBC), blood smear analysis, prenatal diagnostic procedures (amniotic fluid and genetic tests) and molecular tests. Within the CBC examination, one evaluates microcytic anemia. Major thalassemia is characterized by a reduction in Hb levels (<7 g/dl), mean corpuscular volume (MCV) between 50 and 70 fl and mean corpuscular Hb (MCH) between 12 and 20 pg. Thalassemia intermedia is diagnosed by observing a hemoglobin level ranging from 7 to 10 g/dL, MCV between 50 and 80 fl and MCH between 16 and 24 pg. Minor thalassemia is identified through a decrease in MCV and MCH, as well as an elevation in Hb A2 levels.

In individuals with beta thalassemia, a peripheral blood smear reveals morphological changes in RBCs, characterized by microcytosis, hypochromia, anisocytosis, poikilocytosis and the presence of nucleated RBCs. In carriers of beta thalassemia, a decrease in MCV and MCH levels is also observed, along with RBC morphological alterations, albeit not as severe as those in cases of thalassemia major. The pattern of Hb differs across various types of beta thalassemia, with minor individuals exhibiting an increase in HbA2 levels, and homozygous and compound heterozygous patients displaying variable levels.

In β⁰ homozygotes, the beta chain is not synthesized at all, and HbF constitutes approximately 90 to 95% of the total hemoglobin. In β⁺ homozygotes and β⁺/β⁰ types, HbF accounts for 70 to 90% of total blood hemoglobin, while HbA makes up 10 to 30%, contingent upon the extent of beta chain reduction. Quantitative and qualitative analysis of hemoglobin, conducted through cellulose acetate electrophoresis and DE-52 micro chromatography or HPLC, is utilized to ascertain the quantity and type of hemoglobin within the blood. In contemporary times, the molecular methodologies employed for diagnosing beta thalassemia frequently rely on PCR and the identification of common mutations. Among these methods, ARMS and MFPA are the most commonly employed.

ARMS

The Amplification Refractory Mutation System (ARMS) technique is a straightforward approach to identify any type of mutation, particularly point mutations and small deletions. This technique has been established as a gold standard method for diagnosing thalassemia and sickle cell anemia. The fundamental principle behind this method lies in the utilization of polymerase chain reaction (PCR) primers that are specific to a particular sequence, ensuring a precise connection of the 3-end of the primer to the template strand for amplification by PCR. The amplification of the desired fragment is achieved in this method only when the allele with the target sequence is present in the genome. In the case of using ARMS for point mutation detection, two types of complementary primers are designed, one for the mutated allele and another for the normal allele, which are subsequently analyzed for the presence or absence of mutations through PCR, aided by electrophoresis and band observation.

MLPA

The technique known as Multiplex Ligation dependent Probe Amplification (MLPA) is a method characterized by its simplicity, ease of use and high efficiency in the detection of various genetic abnormalities. These abnormalities include changes in DNA copy number, aneuploidies, deletion/duplication mutations and sub telomeric rearrangements. Additionally, the MLPA technique involves the utilization of methylation status. Specifically, this method employs a maximum of 40 different probes that are designed for specific sequences.

Each probe consists of two half-probes, with one of them being marked, and they are connected to the complementary region of their respective sequences in close proximity. Upon complete hybridization of the probes with the target sequence, ligase enzyme facilitates the connection between the halves, and subsequently, the polymerase chain reaction (PCR) is performed. Moreover, MLPA incorporates the use of universal primers, enabling the simultaneous amplification of multiplex PCR for all probes. The execution of the MLPA technique involves five distinct steps, which include:

DNA denaturation and incubation with MLPA probes:

wherein the separation of the DNA strands allows the two halves of the probes to recognize the target sequence. Successful probe connection without any gaps ensures the subsequent steps of ligation and amplification.

Ligation
PCR reaction and fragment amplification
Separation of the PCR products through capillary electrophoresis under denaturing conditions.
Analysis of the results by assessing the peak height or the area of the fluorescence peaks using various software programs like GeneMarker®

TritaGene’s beta thalassemia diagnostic kit for employs the novel Multiple Fluorescent Probe Amplification (MFPA) technique. This technique, in conjunction with specific primers and probes, effectively facilitates the diagnosis of this disease. Notably, the MFPA technique bears close resemblance to MLPA, differing only in the removal of the ligation step.

Beta thalassemia treatment

Individuals with beta thalassemia minor do not require medical intervention. Only when they decide to have children, they can seek guidance from a genetic counselor to obtain suitable measures to avert the transmission of the disease to subsequent generations. Conversely, individuals diagnosed with thalassemia intermedia will encounter instances of mild anemia throughout their lifetime, necessitating blood transfusions on certain occasions. In cases of acute beta thalassemia major and intermedia, diverse treatment modalities may be employed, including but not limited to:

Splenectomy: This procedure is recommended for patients with thalassemia major and intermedia who experience severe hemolysis and consequent splenomegaly due to excessive splenic activity. Splenectomy is recommended when there is a need to inject more than 200-220 ml RBCs/kg with 70% hematocrit, as well as 250-275 ml/kg packed RBCs with 60% hematocrit annually.
Bone marrow transplantation: This method stands as one of the highly effective treatment options for beta thalassemia, necessitating the compatibility of the HLA marker between the donor and recipient.
Blood transfusion: In individuals suffering from beta thalassemia major, blood transfusion is employed to sustain the hemoglobin level in the plasma and treat anemia stemming from endogenous erythropoiesis.