Molecular and Cellular Analysis of a Novel HBA2 Mutation (HBA2: c.94A>G) Shows Activation of a Cryptic Splice Site and Generation of a Premature Termination Codon

Abstract

In this study, we describe the clinical features and provide experimental analyses of a novel point mutation affecting the penultimate nucleotide of the first exon of the HBA2 (HBA2: c.94A>G) gene identified in a 26-year-old female who also carries a heterozygous Hb E (HBB: c.79G>A) variant. The aim of the study was to investigate the impact of this point mutation on the transcriptional activity of the HBA2 gene using a combination of an initial in silico prediction followed by in vitro mutagenesis and transcriptional activity assessment. The analyses revealed that the HBA2: c.94A>G point mutation causes the activation of a cryptic splice site located 49 bp upstream of the exon1-intron1 boundary in both HBA2 long and short isoforms, thus generating a frameshift and a premature termination codon between codons 48 and 49 in the second exon. A rapid degradation of the aberrantly spliced transcripts by the nonsense mediated decay (NMD) surveillance system is highly indicative of an α-thalassemia (α-thal) phenotype. However, the abnormal mRNA may not be entirely degraded since the proband presents a slight splenomegaly that could be the sign of extra vascular hemolysis.

• α-Thalassemia (α-thal)
• Aberrant splice variant
• cryptic splice site
• negative dominant effect
• nonsense mediated decay (NMD)
• premature termination codon
• transition point mutation

Acknowledgements

The authors would like to thank PathWest Laboratory Medicine WA and King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, for providing support for this project.