© 2015 Informa Healthcare USA, Inc. All rights reserved. In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (-55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.