Shen N, Heintz C, Thiel C, Okun JG, Hoffmann GF, Blau N
Molecular Genetics and Metabolism, January 2016
Background: In phenylketonuria (PKU) patients, the combination of two phenylalanine hydroxylase (PAH) alleles is the main determinant of residual enzyme activity in vivo and in vitro. Inconsistencies in genotype-phenotype correlations have been observed in compound heterozygous patients and a particular combination of two PAH alleles may produce a phenotype that is different from the expected one, possibly due to interallelic complementation.
Methods: A dual eukaryotic vector systemwith two distinct PAH proteins N-terminally fused to different epitope tags was used to investigate the co-expression of PAH alleles reported in patients with inconsistent phenotypes. PAH variant proteins were transiently co-transfected in COS-7 cells. PAH activity was measured by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), and protein expression was measured by Western blot. Genotypes were compared with predicted PAH activity from the PAH locusspecific database (PAHvdb) and with phenotypes and tetrahydrobiopterin (BH4) responsiveness from more than 10,000 PKU patients (BIOPKU database).
Results: Through the expression and co-expression of 17 variant alleles we demonstrated that interallelic interaction could be both positive and negative. The co-expressions of p.[I65T];[R261Q] (19.5% activity; predicted 43.5%) and p.[I65T];[R408W] (15.0% vs. 26.8% activity) are examples of genotypeswith negative interallelic interaction. The co-expressions of p.[E178G];[Q232E] (55.0% vs.36.4%) and p [P384S];[R408W] (56.1% vs. 40.8%) are examples of positive subunit interactions. Inconsistencies of PAH residual enzyme activity in vitro and of PKU patients’ phenotypes were observed as well. The PAH activity of p.[R408W];[A300S] is 18.0% of the wild-type activity; however, 88% of patients with this genotype exhibit mild hyperphenylalaninemias (MHPs).
Conclusion: The co-expression of two distinct PAH variants revealed possible dominance effects (positive or negative) by one of the variants on residual PAH activity as a result of interallelic complementation.