Clinical, Biochemical, and Molecular Characterization of HBB Gene Variants in Iraqi Patients with β-Thalassemia Major

β-Thalassemia major is an inherited blood disorder that affects hemoglobin, caused by mutations in the HBB gene. This disease results in chronic hemolytic anemia and dependence on blood transfusions for life. Although the frequency of β-thalassemia major is high in Iraq, there are only a small number of extensive studies that have looked at the relationship between molecular studies, clinical studies, and biochemical studies in this population. The purpose of this study was to identify mutations in the HBB gene of patients diagnosed with β-thalassemia major and to assess the relationships between mutations and both the clinical/biochemical characteristics of patients.  50 transfusion-dependent β-thalassemia major patients and 50 healthy controls. Clinical characteristics were collected for all subjects. All subjects underwent liver function tests, kidney function tests, hemoglobin electrophoresis, and serum ferritin tests. Mutations within the HBB gene were identified using two overlapping polymerase chain reaction (PCR) products, followed by Sanger sequencing, and compared bioinformatic tools. When compared to healthy controls, patients demonstrated significantly higher serum ferritin levels, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and fetal hemoglobin (HbF), with significantly lower HbA1 levels (P < 0.05). Additionally, serum ferritin levels were found to correlate with the transfusion interval, presence of clinical complications, and location of residence. Upon molecular characterization, three genetic variants were identified within the HBB gene: rs1451581925 (386A>T), rs35492035 (59C>G), and rs1609812 (334G>A). Among them, a rare missense variant predicted to replace alanine with proline (p.Ala136Pro) was rs35492035 and rs1609812 were significantly associated with β-thalassemia major susceptibility (P = 0.049) respectively. The metabolic manifestations observed in Iraqi suffering from β-thalassemia major were associated with a set of biochemical alterations and a wide range of HBB gene variants, supporting the influence of and value of rare coding variants to improve the genetic characterization of β-thalassemia in Iraqi   population.