Abstract

Background: Beta-thalassemia is a hereditary hematological illness in which beta-globin chain synthesis is missing or decreased, resulting in inefficient erythropoiesis, persistent hemolysis, and anemia. It is most frequently observed in populations with a high rate of consanguineous marriages; affordability becomes a limiting factor for families with low income. Methods: In this study, we optimized Sanger sequencing of the whole HBB gene covering all three coding exons, two introns, and 5′ and 3′ UTRs. Eleven families were enrolled from Fatimid Foundation Peshawar, Khyber Pakhtunkhwa, Pakistan, with a total of 16 transfusion-dependent patients. An index patient in each family was Sanger sequenced for the HBB gene (n = 11). The variants were classified as per ACMG 2015 guidelines. Results: Sequencing analysis revealed five homozygous pathogenic variations, including three frameshifts: c.27_28dupG (p.Ser10Valfs*14), c.17_18delCT (p.Pro6Argfs17), c.126_129delCTTT (p.Phe42Leufs*19), and two splice-sites (c.92+1G>A, and c.92+5G>C) in nine families. However, two families remained unresolved. The mutation, c.27_28dupG (p.Ser10Valfs*14), was observed in several pedigrees, indicating a probable founder effect or high allele frequency within the affected population. Conclusions: The majority of detected variations in this study were classified as disease-causing, which reside in the first two coding exons and intron-1 of the HBB gene, highlighting its functional importance in the Khyber Pakhtunkhwa population. We recommend performing Sanger sequencing of exon-1-intron-1-exon-2 of the HBB gene with the optimized primers of this study.

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