Molecular Spectrum and Chromatographic- Haematological Characterisation of Alpha Thalassemia Minor (-α/-α), and Alpha Silent Carriers (-α/-αα)

Background and aims Alpha-thalassaemia is a group of disorders characterised by wide phenotypic variation caused by mutations in the α-globin genes (α1 and α2) of chromosome 16. The aim of this study was to investigate the molecular profile of α-thalassemia variants and to compare and characterise the chromatographic behaviour and haematological properties of α-thalassemia minor (-α/-α, --/ααα) and α-silent carriers (-α/ααα) on HPLC. Materials & Methods A dataset of 200 individuals consisting of 42 alpha thalassemia minor (-α/-α, -- /ααα), 103 alpha silent carriers (-α/αα) and 55 normal participants from the Human Genetics Unit (HGU) of the Faculty of Medicine, Colombo, Srilanka was included. Blood samples from each patient were analysed by PCR for genotyping, haemogram and high performance liquid chromatography (HPLC) for characterisation. These data were then comparatively analysed using standard descriptive statistics. Results It was analysed in three sections as haematological, biochemical and molecular. Haematologically, alpha thalassaemia silent carrier was completely normal; alpha thalassaemia minor: decreased haemoglobin level; decreased MCV and MCH, normal RBC count. Alpha Thalassaemia Minor patients show a statistically significant difference from alpha silent carriers and normal population in terms of MCV, MCH, HGB, PCV, RDW. Biochemically, alpha thalassaemia silent carriers were found to have normal alpha-globin chain production, alpha thalassaemia minor: low HBA2 as determined by HPLC. In terms of HPLC tests, similar results were observed between alpha thalassaemia minor patients and alpha silent carriers and between alpha thalassaemia minor patients and the normal group, with no statistical significance for all parameters except HBA2. Molecularly, the most common mutations in both variants are _α3.7 and _α4.2 mutations. Conclusion In summary, despite the haematological and biochemical differences between α-thalassaemia minor and normal individuals, both variants of alpha thalassaemia present a diagnostic conundrum, as CBC and HPLC results for individuals are comparable to normal humans. Although MCV, MCH, HGB, PCV and HBA2 levels differ between alpha thalassaemia minor carriers, alpha silent carriers and the normal group, conventional haemoglobin electrophoresis and haemogram alone have been found insufficient for the diagnosis of alpha thalassaemia. Therefore, although HPLC does not seem to be sufficient to distinguish between normal individuals and the two variants, the decrease in HBA2 levels is an important finding. Identifying a mutated alpha globin gene requires newer molecular diagnostic tests such as next generation sequencing (NGS) and quantitative PCR (qPCR). It should be noted that a given genotype can greatly alter the clinical manifestation by the presence of additional mutations, making the relationship between genotype and phenotype highly variable.