Beta-Thalassemia Gene mutations in Pakistan and NGS for Carrier Status

Introduction

Thalassemia is a hereditary blood disorder characterized by an abnormality in haemoglobin production. It is a major public health concern in Pakistan, with a high incidence and prevalence. This essay delves into the incidence and prevalence of thalassemia in Pakistan, focusing on the genotypes predominantly prevalent and the mutations associated with this disorder.

Incidence and Prevalence

Thalassemia is a significant health issue in Pakistan, and the country has one of the highest rates of thalassemia carriers in the world. The incidence and prevalence of thalassemia in Pakistan can be attributed to various factors, including a high rate of consanguineous marriages, a lack of awareness, limited access to quality healthcare, and a growing population.

1. Consanguineous Marriages: One of the primary reasons for the high incidence of thalassemia in Pakistan is the prevalent practice of consanguineous marriages, where close relatives marry. This increases the likelihood of both parents carrying the same thalassemia gene mutation, resulting in an increased risk of thalassemia in their offspring.
2. Lack of Awareness: Lack of awareness about thalassemia and its inheritance patterns has also contributed to its high prevalence in Pakistan. Many couples are unaware of their carrier status and the risk of passing on thalassemia to their children.
3. Limited Access to Healthcare: Inadequate access to healthcare facilities and genetic counselling services in many parts of Pakistan has hindered efforts to diagnose and manage thalassemia effectively.
4. Growing Population: Pakistan’s growing population exacerbates the burden of thalassemia, as it increases the number of individuals at risk of inheriting thalassemia genes and the demand for healthcare services related to the condition.

Haemoglobin Genes: Structure and Base Pair Composition

Haemoglobin is a crucial protein in our bodies, responsible for transporting oxygen from the lungs to tissues and organs, as well as carrying carbon dioxide back to the lungs for removal. The genes that encode haemoglobin are of particular interest due to their importance in human physiology. In humans, there are two main types of haemoglobin genes: alpha-globin and beta-globin. These genes are found on different chromosomes and play distinct roles in forming functional haemoglobin molecules.

Alpha-Globin Genes:

• Location: Alpha-globin genes are located on chromosome 16
• Total Base Pairs: The alpha-globin gene cluster spans approximately 30,000 base pairs (30 kb).
• Exons: The alpha-globin gene cluster consists of two alpha-globin genes, HBA1 and HBA2, each with three exons. Collectively, the exons in the alpha-globin genes comprise about 450 base pairs.
• Introns: The remaining base pairs within the alpha-globin gene cluster are primarily composed of introns, non-coding sequences that are transcribed but not translated into protein.

Beta-Globin Genes:

• Location: Beta-globin genes are located on chromosome 11.
• Total Base Pairs: The beta-globin gene cluster spans approximately 45,000 base pairs (45 kb).
• Exons: The beta-globin gene cluster includes five genes: HBE1 (embryonic epsilon-globin), HBG2 (gamma-G), HBG1 (gamma-A), HBD (delta-globin), and HBB (beta-globin). The coding exons in these genes, which are translated into haemoglobin protein, amount to approximately 1,500 base pairs in total.
• Introns: Similar to the alpha-globin genes, the beta-globin gene cluster contains introns that make up the bulk of its base pairs.

The regulation and expression of alpha-globin and beta-globin genes are highly controlled and are critical for maintaining the proper balance of different types of haemoglobin throughout an individual’s life. Mutations in these genes can lead to various hemoglobinopathies, such as sickle cell anaemia and thalassemia, which can have significant health consequences.

Understanding the structure and base pair composition of haemoglobin genes is essential for researchers and medical professionals working on diagnosing and treating hemoglobin-related disorders. It also sheds light on the intricate molecular mechanisms underlying oxygen transport and the synthesis of haemoglobin in the human body.

Genotypes and Mutations

Thalassemia can be classified into two main types based on the affected globin chain: alpha thalassemia and beta thalassemia. The specific genotypes and mutations associated with thalassemia vary, but certain mutations are more prevalent in the Pakistani population.

Beta Thalassemia Major (BTM): Beta thalassemia major is the most severe form of beta thalassemia. It is characterized by the complete absence of beta-globin chains. In Pakistan, the most common mutation associated with BTM is the IVS1-5(G→C) mutation. Other mutations such as IVS1-1(G→A) and Codon 8/9 (-AA) are also prevalent.

Beta Thalassemia Intermedia (BTI): Beta thalassemia intermedia is a milder form of beta thalassemia, and its severity varies from person to person. Common mutations associated with BTI in Pakistan include the IVS1-5(G→C) mutation and the IVS1-1(G→A) mutation.

Hemoglobin E-beta Thalassemia (HbE-BT): Hemoglobin E-beta thalassemia is a combination of beta thalassemia and haemoglobin E. In Pakistan, the HbE mutation is often found in combination with beta thalassemia mutations, particularly the IVS1-5(G→C) mutation.

Alpha Thalassemia: Alpha thalassemia results from the deletion or mutation of alpha globin genes. The prevalence of alpha thalassemia is also significant in Pakistan, with various genotypes and mutations. The most common alpha thalassemia mutation is the -α3.7 deletion, followed by the -α4.2 deletion.

Below is a table summarizing prevalent thalassemia mutations in Pakistan, their gene locus, chromosomal number, and the recommended testing methods for detecting these mutations:

Explanation:

• IVS1-5(G→C): This mutation occurs in the HBB gene on Chromosome 11. It can be detected through DNA analysis using PCR-based methods.
• IVS1-1(G→A): Also located in the HBB gene on Chromosome 11, this mutation can be identified through DNA analysis using PCR-based methods.
• Codon 8/9 (-AA): Found in the HBB gene on Chromosome 11, this mutation is detectable through DNA analysis using PCR-based methods.
• -α3.7 Deletion: This mutation involves the HBA1/HBA2 genes on Chromosome 16 and can be detected through DNA analysis using methods like Gap-PCR, MLPA (Multiplex Ligation-dependent Probe Amplification), or PCR-RFLP (Restriction Fragment Length Polymorphism).
• -α4.2 Deletion: Similar to the -α3.7 deletion, this mutation affects the HBA1/HBA2 genes on Chromosome 16 and can be detected through DNA analysis using Gap-PCR, MLPA, or PCR-RFLP.
• Haemoglobin E (HbE): HbE is caused by a mutation in the HBB gene on Chromosome 11. Detection is typically done through haemoglobin electrophoresis, a specialized blood test.
• Haemoglobin H (HbH): HbH results from mutations in the HBA1/HBA2 genes on Chromosome 16. Haemoglobin electrophoresis is used to identify this condition.

Testing for these thalassemia mutations is crucial for carrier screening, prenatal diagnosis, and early detection of affected individuals. Accurate testing enables informed decisions about family planning and provides an opportunity for early intervention and management of the disorder.

Management and Challenges

Managing thalassemia in Pakistan presents several challenges, including:

1. Limited Access to Healthcare: Many thalassemia patients in Pakistan do not have access to specialized medical care, including blood transfusions and iron chelation therapy.
2. Financial Constraints: The cost of managing thalassemia, including regular blood transfusions and medications, can be financially burdensome for many families in Pakistan.
3. Lack of Awareness: Raising awareness about thalassemia, its inheritance patterns, and the importance of carrier screening is essential to reduce the prevalence of the disorder.
4. Genetic Counselling: Access to genetic counselling services is limited in Pakistan, which hinders informed family planning decisions.
5. Blood Donation: Ensuring a consistent and safe supply of blood for thalassemia patients remains a challenge, as voluntary blood donation is not widespread.

Prevention and Future Outlook

Preventing thalassemia in Pakistan requires a multifaceted approach:

1. Public Awareness: Increasing public awareness about thalassemia, carrier screening, and the importance of genetic counselling is crucial.
2. Prenatal Testing: Offering prenatal testing and counselling to at-risk couples can help them make informed decisions about family planning.
3. Blood Donation Drives: Encouraging voluntary blood donation can help maintain a steady supply of blood for thalassemia patients.
4. Access to Healthcare: Expanding access to healthcare services, including specialized thalassemia centres, can improve the management of thalassemia.

Conclusion

Thalassemia is a significant health issue in Pakistan, with a high incidence and prevalence driven by factors such as consanguineous marriages, lack of awareness, limited access to healthcare, and a growing population. The predominant genotypes and mutations associated with thalassemia in Pakistan include beta thalassemia major, beta thalassemia intermedia, haemoglobin E-beta thalassemia, and various forms of alpha thalassemia. Addressing the challenges associated with thalassemia management and prevention is crucial to reducing its impact on the Pakistani population and improving the quality of life for affected individuals.