Molecular Genetic Tests in Thalassemias and the Hemoglobinopathies: Pros and Cons of Different Techniques

Thalassemias and hemoglobinopathies are common inherited monogenic disorders, characterized by defects in the quantity and/or quality of globin chain production. Initial diagnostic evaluation typically includes red cell indices and hemoglobin separation techniques such as HPLC or CZE. Molecular genetic testing becomes necessary to resolve diagnostic ambiguities, guiding prenatal diagnosis, elucidating genotype–phenotype correlations, and occasionally, confirming carrier status. This chapter provides a comprehensive overview of molecular diagnostic techniques used in the evaluation of α-, β-, and rarer forms of the thalassemias. For β-thalassemia as well as most hemoglobinopathies, commonly used methods include allele-specific PCRs (especially ARMS-PCR), DNA reverse dot blot hybridization (RDB), Sanger sequencing, and rarely, MLPA and real-time PCR. For α-thalassemia, multiplex gap-PCR and MLPA are used to detect prevalent gene deletions, while sequencing is increasingly preferred over individual end-point PCRs to identify the less common non-deletional variants. The diagnostic yield, cost-effectiveness, throughput, and ability of these techniques to detect known-versus-novel mutations are discussed. Factors influencing the selection of a diagnostic approach include the local mutational spectrum, laboratory capacity, and clinical scenario. Non-transfusion dependent β-thalassemia (NTDT) genotypes often require assessment of secondary and tertiary genetic modifiers, including co-inherited α-globin gene anomalies and polymorphisms influencing fetal hemoglobin production. There is an emerging role of next-generation sequencing (NGS) for comprehensive genotyping and its integration into high-throughput screening platforms. Recent advances in non-invasive prenatal testing (NIPT), including digital PCR and relative haplotype dosage analysis using cell-free fetal DNA, are transforming reproductive counselling in high-risk couples.