Alpha-Thalassemia

Alpha thalassemia disease

Thalassemia, a prevalent monogenic disease among humans, arises from the diminished production of hemoglobin’s globin chains (α-globin and β-globin). This disease manifests in two major forms, namely alpha and beta thalassemia, although the beta type is clinically more important, but the alpha type is more common. It has been estimated that approximately 5% of the global population are carriers of the alpha thalassemia variant. Hemoglobin, a protein, comprises two constituents, namely Globin and Heme which are responsible for the transport of oxygen from the lungs to the body tissues. Globin, a tetrameric protein present in red blood cells, consists of two alpha polypeptide chains and two beta chains. Each chain encompasses a pocket-like structure wherein a heme group is situated, serving as the site of oxygen binding.

In adults, three main types of normal hemoglobin exist including hemoglobin A, hemoglobin A2, and hemoglobin F with the specific chains within the globin molecule determining the hemoglobin type. Hemoglobin A is constituted by a pair of alpha chains and a pair of beta chains, constituting 95% to 98% of adult hemoglobin. Additionally, there exist other chains like zeta, gamma, epsilon and delta chains that have been involved in the evolution of globin structure from the embryo to the adult stage in humans. The table presented below illustrates the various types of normal hemoglobin found in adults, along with their respective proportions.

Normal Hemoglobins

HbH disease

Hb Bart syndrome

Normal

Composition

Hemoglobin Type

60%-90%

95%

α₂β₂

Hemoglobin A

<2.0%

<2.5%

α₂δ2

Hemoglobin A2

<1.0%

α₂γ₂

Hemoglobin F

2%-5%

85%-90%

γ₄

Hb Bart

0.8%-40%

β₄

HbH

10%-15%

ζ2γ2

Hb Portland

Genes that encode globin components are located on distinct chromosomes within a cluster. The HBA1 gene positioned on chromosome 16 (16p13.3), while the HBB gene located on chromosome 11 (11p15.4). Moreover, the MB gene is located at 22q12.3, the CYGB gene is positioned at 17q25.1 and the NGB gene is found at 14q24.3. Within the genome, the MB, CYGB and NGB genes exist as singular copies, whereas the HBB and HBAs genes are clustered and associated with several other genes.

It is noteworthy that all of these genes consist of at least three exons that are separated by introns. Individuals who suffer from alpha thalassemia exhibit a defect in the synthesis of the alpha chain. If a mutation occurs and prevents the production of one of the chains, it results in alpha thalassemia minor. On the other hand, if the production of both alpha chains is disrupted, alpha thalassemia major is manifested.

Types of alpha thalassemia

Clinically, alpha thalassemia has two forms which are:

Alpha thalassemia patient conditions:

Hb Bart syndrome

Another term for this particular type of alpha thalassemia is known as Bart hydrops fetalis syndrome, which is regarded as the most severe manifestation of this disorder (thalassemia major) and frequently results in mortality during infancy. The hemoglobin composition of infants affected by Hb Bart is predominantly composed of γ4, which possesses minimal physiological functionality.

Within this specific variant of alpha thalassemia, only fetal Portland hemoglobin (ζ2γ2) (Hb Portland) exhibits functional activity and serves as the determining factor for the survival of newborns. Fetuses afflicted by Barrett syndrome generally succumb either within the womb (between the 23rd and 38th week of gestation) or shortly after delivery.

Cause:

The main cause of this disease is the occurrence of large deletion/inactivation mutations in all four α-globin coding genes –/– (deletion/inactivation of all four α-globin genes), which includes HBA1 and HBA2 genes.
Rarely, non-deletion variant (–/αND) is observed Bart syndrome.

Symptoms:

The beginning of general edema of the body before birth
Severe anemia
Enlargement of liver and spleen (Hepatosplenomegaly)
Cardiovascular diseases
Abnormalities of the genitourinary system
Extramedullary erythropoiesis
Massive placenta
Variable reticulocytosis that may be more than 60%
delay in brain growth
Skeletal abnormalities

HbH disease

Hemoglobin H disease, the most severe non-fatal manifestation of alpha thalassemia, is characterized by the presence of a solitary functional copy of the HBA gene, leading to the manifestation of mild to moderate anemia in newborns. In this particular pathologic condition, individuals display a hemoglobin configuration consisting of 4β subunits, thus rarely necessitating blood transfusions.

Cause:

The main cause of this disease is a large deletion/inactivation mutation in 3 α-globin genes (–/-α)
Rarely, HbH disease is caused by compound heterozygous for a deletion in the MCS-R2 gene and an additional deletion in the alpha gene [(αα)MCS-R2/-α]

Symptoms:

Spleen and liver enlargement (Hepatosplenomegaly) in most patients
Slight jaundice
The possibility of gallstones or acute periods of hemolysis in response to infections or exposure to oxidant drugs.
Microcytic anemia
bone changes
Moderate reticulocytosis about 3% to 6%

Carrier modes of alpha thalassemia

Alpha-thalassemia trait

In the situation of alpha-thalassemia trait, an individual possesses two healthy copies of the alpha globin gene. This can be observed as sis with the -/αα genotype or trans with the -α/-α genotype. The carriers within this group exhibit a relatively normal structure of hemoglobin. However, they do display mild levels of hypochromic-microcystic anemia, also known as microcytic hypochromic trait.

The risk of transmitting the disease to the next generation in the sis mode is as follows:

25% HbH disease
25% off carrier
25% α +-thalassemia trait/carrier
25% of normal hemoglobin

The risk of disease transmission to the next generation in trans mode:

50% silent carrier
50% α +-thalassemia trait/carrier

α-Thalassemia Silent Carrier

In the case of α-thalassemia silent carrier, there is a deletion of only one alpha globin gene, resulting in the α-/αα genotype for the carrier. Hemoglobin synthesis remains predominantly normal and functional in the body in this case. This particular type of alpha thalassemia can arise from non-deletion mutations and does not present any distinct clinical symptoms in the affected individual.

The risk of transmitting the disease to the next generation is as follows:

25% risk of HbH disease
25% off carrier
25% α +-thalassemia trait/carrier
25% of normal hemoglobin

Carrier State Terminology	Definition	Symbol	Genotype	name
α-thalassemia silent carrier	Some α-globin protein is produced from one chromosome 16	α⁺	-α/αα	α-thalassemia silent carrier
α⁰ trait (α⁰-thalassemia)	Zero α-globin protein is produced from one chromosome 16	α⁰	–/αα	α-thalassemia trait/carrier (sis)
α+-thalassemia trait	Some α-globin protein is produced from each of two chromosomes 16	α⁺	-α/-α	α-thalassemia trait/carrier (trans)

Genetic diseases associated with alpha thalassemia

In this particular section, we shall introduce several genetic disorders that are associated with alpha thalassemia, namely:

Alpha thalassemia / intellectual disability syndrome, related to chromosome 16 (ATR-16 Syndrome)

This specific genetic anomaly is characterized by a contiguous gene deletion syndrome resulting from a substantial terminal deletion of the short arm of chromosome 16 at position 16p13. This rare genetic disorder leads to individuals experiencing monosomy as a consequence of losing a significant portion of chromosome 16 (approximately 1700-900 kb) as well as several adjacent genes, including the HBA1 and HBA2 genes.

It is important to note that this deletion mutation occurs spontaneously without any familial history of the disease. Among the symptoms exhibited by these patients, one can observe intellectual disability, club feet, microcephaly, alpha thalassemia, hypertelorism, small ears, short neck and a broad, prominent nose bridge.

Acquired alpha thalassemia (α-thalassemia-myelodysplastic syndrome; ATMDS)

On rare occasions, individuals who previously had normal erythropoiesis may develop alpha thalassemia, typically due to hematologic malignancy. It is worth mentioning that all cases of acquired alpha thalassemia are associated with an underlying clonal disorder of hematopoiesis, most commonly a form of myelodysplastic syndrome (MDS). Consequently, this condition is now recognized as alpha thalassemia myelodysplasia. The clinical manifestations of ATMDS include the severe form of HbH disease with hypochromic microcytic anemia.

The etiology of alpha thalassemia and its mode of inheritance

Alpha thalassemia syndrome is correlated with varying degrees of mutations in the genes responsible for coding alpha globin. In a healthy individual, there exist four alpha globin genes, all situated at the position 16p13.3. The cluster of alpha globin genes comprises a total of 7 genes, with the most significant ones being the HBA1 and HBA2 genes. The occurrence of mutations and subsequent inactivation of these genes results in the cessation of alpha globin gene expression and the absence of product synthesis. Hence, as the number of mutated genes increases, the severity of the disease escalates. Notable deletion mutations that transpire in the alpha globin gene include -α^3.7 (3.7-kb deletion), -α^4.2(4.2-kb deletion), —^SEA (Southeast Asian deletion), —^THAI (Thailand deletion), —^FIL (Filipino deletion), —^MED (Mediterranean deletion), -α^CD59, -α^20.5and -α^{IVS I-1}as indicated.

Amongst other mutations that induce alpha thalassemia in individuals, it is worth noting the existence of non-deletion mutations in the alpha globin gene. These mutations are accountable for diseases such as Hb Constant Spring (CS). The aforementioned form of alpha thalassemia is triggered by a substitution mutation in the termination codon TAA→CAA. Another variation of this particular type of alpha thalassemia is Hb Pakse (-α4PS), which experiences a substitution mutation in the end codon (UAA→CAA). This group of mutations that arise in the termination codon result in the extension of the alpha globin protein and the emergence of various clinical complications, including liver failure, heart diseases and endocrine disorders.

In certain patients with Hb Constant Spring (CS), when the hemoglobin level experiences a drastic reduction, red blood cell transfusion becomes necessary. Additionally, among other mutations that lead to an alteration in the production of alpha globin, there exists the triplication of α-globin genes (ααα) or α-triplication. This phenomenon is caused by homologous recombination between duplicated α-globin genes. Individuals with this mutation rarely exhibit clinical symptoms and their blood parameters appear to be normal.

However, when α-triplication in the alpha-globin genes coexists with a mutation in the β-globin gene in an individual, it plays a significant role in the pathophysiology of thalassemia by exacerbating the imbalanced synthesis of the alpha-globin chain and impacting erythroid maturation and survival. There are two types of α-triplication in the alpha globin gene, namely ααα^anti3.7(predominant in Africa, the Middle East and the Mediterranean) and ααα^anti4.2(common in Asia).

In addition, a remarkable reorganization of the cluster of alpha globin genes, known as the HKαα allele (αα Hong Kong), has been documented. This particular allele exhibits crossover junctions between -α^3.7and ααα^anti4.2. It is important to mention that the HKαα allele does not truly possess three copies of the alpha globin gene and is not regarded as a variant of α-triplication.

Prevalence rate of alpha thalassemia

The prevalence of Alpha thalassemia, a hereditary condition, is observed on a global scale; however, it is particularly prominent among the Asian population. In specific regions of Southeast Asia and China, approximately 40% of carrier population exhibit symptoms of this disease. Additionally, other populations with a notably elevated incidence of alpha thalassemia include those residing in the Middle East, India, Africa and Mediterranean areas. The subsequent table outlines the most frequently observed mutations of the alpha globin gene in individuals affected by alpha thalassemia across diverse populations.

Common type oh alpha thalassemia mutation	population
-α^3.7	Equatorial Africa includes countries such as Nigeria, Ivory Coast and Kenya
-α^3.7/-α^3.7	Mediterranean
-α^3.7	Oman
-α^3.7 / -α^4.2	India
· Alpha⁰-thalassemia alleles (–^SEA, —^THAI, —^FIL) · α⁺-thalassemia alleles (-α) · Hb^{Constant Spring} (Hb^CS)	South East Asia

Other names for alpha thalassemia

Alpha-thalassemia
Α-thalassemia

Diagnosis methods of alpha thalassemia

In this section, we will introduce various potential approaches for the diagnosis of alpha thalassemia disease in individuals, which include:

Blood hemoglobin analysis

Determining the quality of hemoglobin in individuals’ blood can be ascertained through the utilization of techniques such as High Performance Liquid Chromatography (HPLC) and electrophoresis. These methodologies are frequently employed to identify the specific type of thalassemia and to determine whether individuals are carriers.

Complete blood count

A comprehensive blood count (CBC) examination is conducted within a laboratory setting using automated hematology analyzers. However, hemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) are unable to differentiate between thalassemia trait and iron deficiency, as well as between alpha and beta thalassemia. Moreover, thalassemia does not directly impact platelet and white blood cell (WBC) counts. Individuals with thalassemia exhibit an elevated red blood cell (RBC) count, which serves as a distinguishing characteristic compared to iron deficiency anemia, where a low red blood cell count is observed.

Blood iron check

If the level of serum ferritin is within the normal range or slightly increased, while the amount of transferrin remains normal, it signifies the presence of thalassemia. Conversely, in cases of iron deficiency anemia, ferritin levels decrease and transferrin levels increase.

Peripheral smear

The examination of a peripheral blood smear allows for the assessment of microcytic hypochromic anemia, target cells, teardrop cells and cells with basophilic stippling. However, these findings are commonly associated with iron deficiency anemia, whereas the presence of “golf ball-like hemoglobin inclusions” indicates the presence of HbH disease.

Genetic test

One of the challenges in the diagnosis and treatment of alpha thalassemia disease is the presence of various types of deletion/non-deletion mutations in certain patients, each causing distinct symptoms. A highly effective approach to accurately diagnose alpha thalassemia disease involves the utilization of diverse genetic tests, such as MLPA (multiplex ligation-dependent probe amplification), Reverse Dot Blot, NGS, melting curve analysis with Real-Time PCR, and Allele-specific PCR.

The MLPA technique (Multiplex ligation-dependent Probe Amplification)

It is a type of Multiplex PCR that is used to detect duplication/deletion mutations in a specific region. This technique utilizes primers and labeled probes specifically designed to identify certain sequences. The labeled probes utilized in this method are synthesized in the form of two half-probes, namely the 5′ MLPA probe and the 3′ MLPA probe, both serving the purpose of identifying specific sequences.

In addition, universal primers are employed in MLPA, allowing for simultaneous multiplex PCR amplification of all probes. To address the identification of deletions and duplications within the human alpha globin gene cluster, including -α^3.7, -α^4.2, —^SEA, —^THAI, —^FIL, —^MED deletions, -α^20.5 and the occurrence of α-triplication associated with the development of alpha thalassemia disease, the Trita® α-thalassemia InDel Diagnostic Kit has been developed. This kit functions based on the novel Multiple Fluorescent Probe Amplification (MFPA) method, enabling the diagnosis of alpha thalassemia through the utilization of DNA derived from the peripheral blood of adults or infants.

Reverse Dot Blot analysis

This technique is used to better identify suspected mutations, where the PCR product is transferred on a membrane filter plate in the form of a dot (Blot) and hybridized with an allele-specific oligomer (ASO) DNA probe.

NGS technique (Next Generation Sequencing)

In NGS technique or next generation sequencing, the whole genome of a person can be sequenced at high speed, which is used in the analysis of all alpha globin gene coding regions, key regulatory regions and modifier genes.

Direct DNA Sequencing

In this method, the PCR product is directly sequenced or read using the Sanger technique.

Melting curve analysis with Real-Time PCR

Real-Time PCR method can be considered better than conventional PCR method because less time is spent on the amplification reaction process, also the risk of contamination after PCR is low.

Alpha thalassemia treatment

Individuals who carry alpha thalassemia (-+α or -α0) generally do not necessitate medical intervention as their anemia tends to be exceedingly mild in nature. Naturally, it is crucial to meticulously address the potential occurrence of anemia resulting from concurrent nutritional deficiencies, such as iron, folate or vitamin B12 scarcity. In such cases, proper management is of utmost importance. Administering iron supplements to a non-anemic individual may lead to an increase in blood iron accumulation, thereby posing a significant risk. Consequently, the consumption of iron tablets must be conducted under the careful oversight and discretion of a medical professional.

Patients diagnosed with alpha thalassemia type HbH may occasionally exhibit slight symptoms that do not warrant blood transfusions; however, in certain instances, severe symptoms manifest, necessitating regular transfusions. If hemoglobin (Hb) levels undergo an abrupt decline due to hemolytic or aplastic crises, individuals afflicted with HbH knockout disease may require red blood cell transfusions. The gravity of Hb Bart’s alpha thalassemia is such that, in the majority of pregnancies where the fetus is affected by this condition, the fetus is terminated, resulting in the termination of the pregnancy. Nonetheless, there have been instances where a number of fetuses with this condition have survived and been born; statistics indicate that approximately 50% of these infants experienced growth retardation, while 20% exhibited neurodevelopmental delay.