Complex interaction of δ-globin variant HbA2-Famagusta combined with β-globin variant Hb Monroe in cis with the HBB:c.-92C>G mutation: hematological characteristics and molecular diagnostic insights

Hemoglobin (Hb) variants can cause clinical conditions like thalassemia. Understanding their molecular phenotypes and clinical profiles is essential for accurate diagnosis and genetic counseling. Blood samples from a 67-year-old Thai female and four relatives were analyzed using HPLC and capillary electrophoresis. PCR techniques and sequencing were used to identify globin gene mutations and β-globin haplotypes. Pathogenicity and transcription factor-binding sites were predicted. A rapid diagnostic method was developed and validated using 1,414 samples to assess the geographic distribution of the variants. Hb analysis revealed HbA, HbA2, and a slower-migrating Hb fraction. Mutations identified included HBB:c.92 G > C(HbMonroe) in cis with HBB:c.-92C > G, and HBD:c.60C > A(HbA2-Famagusta). This novel combination exhibited normal HbA2 levels, potentially masking β-thalassemia. Combining HbMonroe with HbE leads to severe clinical symptoms. HbA2-Famagusta was predicted to be benign, while HbMonroe was deleterious. The geographic distribution of HbMonroe was found to be 0.09% with an allele frequency of 0.00042 and 1.30% with an allele frequency of 0.00649 in β-thalassemia carriers and EF syndrome, respectively. HbMonroe is a β0-thalassemic variant affecting splicing and structure, undetectable by standard Hb analysis. Including HbA2 variants in total HbA2 quantification is essential for accurate diagnosis and improved genetic counseling in thalassemia-endemic areas.