Research Details

Hemoglobin (Hb), the main protein in red blood cells, carries oxygen throughout the body. Adult hemoglobin (Hb A) consists of two alpha and two beta globin chains, produced by HBA1/HBA2 (chromosome 16) and HBB (chromosome 11) genes [1]. Mutations in HBB genes or their regulatory regions can reduce or halt globin chain production, leading to beta-thalassemia, a disorder marked by microcytic hypochromic anemia and impaired oxygen transport [2-5]. Current diagnostic tools for beta-thalassemia include capillary electrophoresis and fragment sequencing [6-8], which covers only the structural region of the beta-globin (HBB) gene. The promoter region which regulates the activity of the HBB gene and may contribute to beta-thalassemia trait is not covered by existing diagnostic methods.

Promoter regions of HBB genes evolve rapidly, with higher nucleotide substitution rates than introns [9]. Multiple Single Nucleotide Polymorphisms (SNPs) have been identified in the alpha and beta globin gene promoters [10,11] and numerous other studies, some of which influence promoter activity, gene regulation, and disease susceptibility [12,13]. Many functional mutations cluster between +50 to -500 from the transcription start site, though others occur further upstream [14]. Distinguishing between polymorphic and pathogenic variants is crucial for accurate clinical interpretation [10]. 

There were previously reported mutations in the promoter region of HBB. Cis-regulatory elements described include CACCC boxes, CCAAT box, TATA box which were both hypothesized to result in beta-thalassemia or even “silent” thalassemia. A study in the Spanish population identified six mutations in the proximal and distal regions of HBB gene promoter affecting CACCC and TATA boxes causing moderate beta-thalassemia and there were also silent mutations where carriers have normal blood parameter indices [13]. In the Chinese population, carriers of a novel –73 (A→T) CCAAT-box had a completely silent β-thalassemia phenotype despite one compound heterozygote child showing microcytosis. In another study where ~192,000 individuals were screened, a novel 5′-UTR (cap region) mutation c.23A>G was found with 75 carriers (≈0.0389%) but there were no noticeable hematologic parameters between carriers and controls [15].

Other mutations such as substitutions in HBB gene promoter were reported to present as normal or borderline in specific complete blood count (CBC) parameter including red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin (HGB) [16-18]. These mutations showing a hematologic picture without microcytosis and only borderline for other β⁺thalassemia indicators make routine screening ineffective. Moreover, these findings reinforce that promoter variants in HBB gene promoter can produce very mild or silent effects on blood indices. These mutations in the regulatory region of the HBB gene can be overlooked in conventional diagnosis which poses risks with possibility of additive impact from independent assortment. 

This study was conducted to evaluate the potential of mutations within the HBB gene promoter by analyzing associated HBA2 levels, HBB gene expression profiles and complete blood count (CBC) parameters among participants. Moreover, the identification of potentially novel promoter variants may provide additional evidence supporting the clinical relevance of regulatory-region mutations in β-thalassemia. The findings of this study may contribute to improving current diagnostic strategies by emphasizing the importance of including the HBB promoter region in routine molecular screening for thalassemia, particularly in the cases of mild, borderline, and silent hematologic phenotypes.