Genetic Approach in Diagnosis and Follow-Up of Patients with Thalassemia: A Comprehensive Narrative Review

Thalassemia represents the world’s most prevalent inherited hemoglobin disorder, affecting approximately 4.4 per 10,000 live births globally. Accurate genetic characterization is indispensable both for definitive diagnosis and for lifetime clinical monitoring. The past two decades have witnessed a paradigm shift from conventional protein-based assays toward comprehensive molecular techniques, including next-generation sequencing (NGS) and third-generation (long-read) sequencing, which in turn have enabled reproductive applications such as preimplantation genetic testing for monogenic disease (PGT-M) to identify unaffected embryos before implantation. (1) To systematically evaluate the molecular techniques available for confirming the diagnosis of alpha- and beta-thalassemia, including their diagnostic accuracy, indications, and limitations; (2) to examine how genotype–phenotype correlation and genetic modifier profiling inform clinical prognosis and therapeutic decision-making; and (3) to define evidence-based genetic monitoring parameters for longitudinal follow-up of patients receiving transfusions, iron chelation, and novel curative therapies including gene therapy. A comprehensive narrative review was conducted by systematically searching PubMed/MEDLINE for English-language peer-reviewed articles published between January 2000 and December 2024. Forty-three studies were ultimately included after applying predefined inclusion and exclusion criteria. Quality of included studies was assessed using SANRA (Scale for the Assessment of Narrative Review Articles). HPLC and capillary electrophoresis remain first-line phenotyping tools; DNA-based confirmation is mandatory for complete genotyping. Among known, previously characterized mutations, NGS-based targeted panels achieve > 95% detection sensitivity, but they require MLPA co-testing or long-read sequencing to detect structural variants such as large deletions. Genotype–phenotype prediction is substantially improved, though not rendered fully deterministic, by profiling three major modifier loci: XmnI (Gγ), BCL11A, and HBS1L-MYB. PGT-M using NGS achieves near-complete genotyping accuracy (>99%) with live birth rates of 40–60% per frozen embryo transfer cycle. For patients receiving curative gene therapy (exagamglogene autotemcel/Casgevy), molecular follow-up protocols spanning 15 years are now recommended. Cardiac T2* MRI remains the most reliable non-invasive tool for iron overload follow-up, superior to serum ferritin alone. A tiered, genotype-informed approach—combining HPLC/CE phenotyping, targeted molecular diagnostics, genetic modifier profiling, and periodic re-evaluation—optimizes diagnostic precision and guides individualized management across the thalassemia spectrum. Integration of PGT-M and long-read sequencing into standard care pathways, alongside robust gene therapy follow-up protocols, will define the next era of thalassemia genetics.