title: Co-expression of phenylalanine hydroxylase variants and effects of interallelic complementation on in vitro enzyme activity and genotype-phenotype correlation.
abstract: 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 system with 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 locus-specific 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 genotypes with 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Dietmar-Hopp Metabolic Center, University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany.
authors.forename: Nan
authors.initials: N
authors.lastname: Shen
authors.affiliation: Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, United States.
authors.forename: Caroline
authors.initials: C
authors.lastname: Heintz
authors.affiliation: Dietmar-Hopp Metabolic Center, University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Thiel
authors.affiliation: Dietmar-Hopp Metabolic Center, University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany.
authors.forename: Jürgen G
authors.initials: JG
authors.lastname: Okun
authors.affiliation: Dietmar-Hopp Metabolic Center, University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany.
authors.forename: Georg F
authors.initials: GF
authors.lastname: Hoffmann
authors.affiliation: Dietmar-Hopp Metabolic Center, University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany. Electronic address: nenad.blau@med.uni-heidelberg.de.
authors.forename: Nenad
authors.initials: N
authors.lastname: Blau
mesh_terms: D000483:Alleles; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D005260:Female; D056726:Genetic Association Studies; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D010641:Phenotype; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D010661:Phenylketonurias / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011993:Recombinant Fusion Proteins; D001708:Biopterins; D010651:Phenylalanine Hydroxylase; C003402:sapropterin
keywords: Hyperphenylalaninemia; PKU; Phenylketonuria; Tetrahydrobiopterin