Functional analysis of 6 variations in

Objective: We aimed to investigate the functional alterations caused by pathogenic variants in the Methods: This study is an experimental research with a duration from January to September 2022. We selected six variants for analysis, including a double missense variant, c.150C>G (p. Asp50Glu) and c.326A>T (p. Asn109Ile); three deletions, c.411_412del (p. Met137Ilefs101), c.533_542del (p. Val178Alafs90), and c.684delA (p. Ala229Leufs43); a nonsense variant, c.214G>T (p. Glu72); and a duplication, c.663_692dup (p. Ala225_Ala234dup). We constructed expression vectors containing these variants and transfected them into HeLa cells. Confocal microscopy was used to observe the subcellular localization of the expressed proteins. We evaluated gene expression using dual luciferase reporter assays and quantitative PCR. Results: Proteins expressed by vectors with deletion variants were predominantly localized to the nucleus, while those with the double missense variant exhibited diffuse expression throughout the cell. Proteins from nonsense and duplication variants localized to the cytoplasm. Luciferase activity assays revealed that proteins encoded by the p. Ala229Leufs43, p. Glu72, and p. Ala225_Ala234dup variants significantly diminished the inhibitory effects on the transcription of the Conclusions: The double missense variant does not exert a superimposed inhibitory effect on gene expression. Despite differences in subcellular localization, all mutant proteins produced by these variants likely interfere with downstream gene expression through a shared pathway. Furthermore, mutant