[ expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase].

Objectives: To study the expression of three phenylalanine hydroxylase (PAH) mutants (p.R243Q, p.R241C, and p.Y356X) and determine their pathogenicity.

Methods: Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein. Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells. Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants, and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay.

Results: Bioinformatics analysis predicted that all three mutants were pathogenic. The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control (>0.05), while the mRNA expression level of the p.Y356X mutant significantly decreased (<0.05). The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control (<0.05). The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control (<0.05), while there was no significant difference between the p.R243Q mutant and the wild type control (>0.05).

Conclusions: p.R243Q, p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells, with p.R241C and p.Y356X mutants also affecting the function of PAH protein. These three PAH mutants are to be pathogenic.