Molecular basis of phenylketonuria in Venezuela: Presence of two novel null mutations

Abstract

This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela. In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe. In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3. The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems. This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients.

Expression Analysis of Phenylketonuria Mutations

“…PAH with the S349L mutation produced an unstable protein undetectable in SDS-PAGE. The expression of this mutation in eukaryotic cells showed that the mutant enzyme was not detected by Western blot, and consequently no residual activity in bacteria nor in COS cells could be measured (17). In this work we have extended the expression analysis to two other mutations, L348V and V388M, which have been previously reported to retain residual activity in COS cells, 25-33% for L348V (3) and 43% for V388M (16).…”

“…As described in a preliminary report, we have overexpressed and affinity purified PAH protein as fusion protein with MBP (17). PAH with the S349L mutation produced an unstable protein undetectable in SDS-PAGE.…”

“…Expression analysis was performed in COS cells using the pRc/CMV expression vector (Invitrogen), as described previously (16), and in E. coli using pMALc2 (Biolabs) where PAH is cloned as a fusion protein with MBP under the control of an inducible promoter (17,18). Mutations were introduced in the PAH cDNA sequence by site-directed mutagenesis using the Gene Editor kit from Promega.…”

“…Bacteria were harvested 16 -21 h after induction and disrupted by sonication in Na-Hepes 20 mM, NaCl 0.2M, pH 7.0, with 1 mg/ml lysozyme and 0.2 mM Pefabloc. After centrifugation, the supernatant (crude protein extract) was used to measure PAH residual activity by monitoring conversion of 14 C-Phe to 14 C-Tyr (17). Briefly, the standard reaction mixture performed in a 50-l final volume contained 150 -300 g of total protein, catalase (10 l at a concentration 100 units/l), 14 C-Phe (0.5 Ci, Ͼ450 mCi/mmol), and 10 l of 1 mM 6-methyltetrahydropterin (synthetic cofactor, added last), in 20 mM Na-Hepes, 0.2 M NaCl, pH 7.…”

“…This apparent catalytic effect is discrepant with the fact that V388M affects residues located outside the active site in the three-dimensional structure of the PAH enzyme (13). On the contrary, S349L is located lining the active site, but expression both in COS cells and in Escherichia coli rendered a highly unstable protein (17). To clarify and extend these results, we have used several complementary expression systems (eukaryotic and prokaryotic) and experimental conditions (co-overexpression with chaperonins, different growth temperatures), providing evidence that the mutations affect directly the folding and stability of the protein.…”