Identification of a Point Mutation in Position – 83 (G&gt;A) of the β Globin Gene Promoter and the Influence of Globin Genes Mutation on the HbA2 Level
Authors: Cadet Estelle , Foulon Karine , Claisse Jean-Francois , Rochette Jacques 
Published: April 2020

Carriers of typical β thalassemia alleles have microcytic hypochromic red blood cells with elevated Hb A2 values and a normal or slightly elevated level of Hb F. The increased level of Hb A2 is a reliable marker for heterozygous beta-thalassemia while Hb A2 levels within normal ranges do not exclude heterozygous beta-thalassemia. A male patient of Gabonese ancestry with both parents belonging to the Obamba sub-population was referred to us because of persistent microcytosis with anisopoikylocytosis and spherocytosis in the absence of clinical signs. The Hb separation pattern on alkaline electrophoresis and on ion exchange HPLC was normal. The Hb A2 level was 3.5% and the HbF level was 0.8%. Direct sequencing of the β globin genes revealed a G&gt;A transition in position − 83 upstream of the cap site. The − 83 G&gt;A substitution is located in a region between the CACCC motif (from − 90 to − 86) and the CCAAT motif (from − 76 to − 72) and has not been reported before. An alpha thalassemia associated with the β mutation was considered as well as a 𝛉 gene defect. Both hypotheses were based on the Hb A2 level.

The Gap-PCR revealed the presence of − α3.7 (rightward) deletion in the heterozygous state in the proband while the 𝛉 genes did not show any mutation when compared to the normal sequence. In some instances, carriers for β thalassemia mutation may have normal Hb A2 level and can be mistaken for α-thal and/or 𝛉-thal or iron deficiency. We have revisited the Hb A2 level in different genotype conditions by using the VARIANT TM HPLC system with the β-Thalassemia short program (Bio-Rad Laboratories, Hercules, CA, USA). We have established thal carriers of typical nondeletional β-Thal alleles with no alpha thalassemia and in the absence of iron deficiency have a remarkable uniform level of Hb A2, rarely more than 6% and with an average value of 4.82 % ± 0.67( n=214). Point mutations located within the β globin gene promoter gives rise to an average of Hb A2 level of 5.42% ± 0.76 (n= 27). When a premature termination codon is present the Hb A2 percentage reaches 5.90 % ± 0.32 (n=12). The Hb A2 level drops to an average of 2.54 % ± 0.40 (n=108) in individuals carrying the − α3.7/αα genotype only. The association of the − α3.7/αα genotype with a non deletional β thalassemic allele shows an average of Hb A2 at 3.30 % ± 0.32 (n=134). Thus we explain the normal Hb A2 level in our patient by the co-inheritance of the β mutation together with the common heterozygous deletional α thalassemia (− α3.7/αα). Also, it is very unlikely that the − α3.7/αα genotype by its own may explain the red cell indices found in our patient (MCV=73 ; Hb = 12g/dl ; MCH = 23 pg) Co-inheritance of α and β thalassemia should always be considered when non iron deficient hypochromic microcytic anemia is diagnosed together with normal Hb A2 level, especially in regions with a high prevalence of both types of thalassemia. DNA analyses are needed for accurate diagnosis as this combination of genotypes can have implications for genetic counselling and prenatal diagnosis.