The current genetic investigation was conducted to characterize the molecular basis of variable ocular abnormalities observed in a consanguineous Pakistani family affected by β-thalassemia. Peripheral blood samples were collected from affected and unaffected members of the family, followed by targeted Sanger sequencing of the HBB gene to identify potential pathogenic variants. Comprehensive in silico analyses were carried out was also done. Clinical and laboratory findings were consistent with a severe transfusion-dependent anemia. Genetic screening revealed a homozygous insertion mutation, c.27_28insG, resulting in a frameshift and premature stop codon at position p.Ser10Valfs*14. This mutation was found to be associated with the β⁺/β⁺ thalassemia major phenotype in the two affected siblings. Interestingly, both individuals exhibited diverse and progressive ocular manifestations, including congenital cataract, high-grade progressive myopia, involuntary eye movements (nystagmus), and bilateral strabismus, suggesting a possible correlation between the thalassemic genotype and retinal dysfunction. The computational analyses demonstrated significant structural perturbations in the mutated β-globin protein, supporting the pathogenic nature of the frameshift variant. Furthermore, ophthalmological evaluation, particularly fundoscopic examination, confirmed the presence of structural retinal abnormalities, which may be a secondary consequence of chronic hypoxia or iron overload commonly seen in thalassemia major. This study not only expands the phenotypic spectrum associated with β-thalassemia but also highlights the potential link between thalassemic mutations and extra-hematological complications such as ocular pathology. Early molecular diagnosis and clinical intervention could significantly improve disease management and reduce the burden of preventable complications in future generations.

Keywords: Mutation, Pakistan, Retinal, Visual, Phenotype, HBB gene.