In vitro expression study of novel mutations in phenylalanine hydroxylase gene.
- Author:
Xin-shun ZHANG
1
;
Xue-fan GU
;
Li-li LIANG
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Animals; COS Cells; Cercopithecus aethiops; Genetic Association Studies; methods; Humans; Molecular Sequence Data; Mutation, Missense; Phenylalanine Hydroxylase; genetics; Sequence Alignment
- From: Chinese Journal of Medical Genetics 2013;30(6):673-677
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo study the in vitro expression of 6 novel missense mutations (R270G, P275A, F121L, A156P, E183G, I324N) and a previously described R408Q mutation of phenylalanine hydroxylase (PAH) gene and explore the genotype-phenotype correlation through comparison of protein levels and residual enzyme activities.
METHODSSeven expression vectors containing PAH cDNA were constructed with a site-directed mutagenesis kit. The plasmids were extracted and sequenced to confirm the target mutations. pcDNA3.0 containing PAH cDNA was transfected into COS-7 cells and total proteins were extracted 48 h after transfection. The quantities of proteins and residual enzyme activities of the 7 mutants were assessed with the wild-type PAH gene as reference.
RESULTSRelative quantities of PAH proteins for R270G, P275A, F121L, A156P, E183G, I324N and R408Q were 10.5%, 56.6%, 54.3%, 8.7%, 8.5%, 67.3% and 85.4%, respectively. The residual enzyme activities were 7.7%, 27.6%, 19.0%, 10.4%, 9.1%, 50.6% and 40.2%, respectively.
CONCLUSIONPAH residual enzyme activities of 7 PAH mutants were all significantly reduced.
