Mutation analysis of the phenylalanine hydroxylase gene using heteroduplex analysis with synthetic DNA constructs

Tyfield, Linda; Stephenson, A; Bidwell, J. L.; Wood, Natalie J.; Cockburn, F.; Harvie, Ann; Smith, Iain

doi:10.1111/j.1651-2227.1994.tb13450.x

Cited by 3 publications

(2 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, in the N167D mutant tetramer the repulsive electrostatic interaction between the negatively charged carboxyl groups of Asp 167 and Asp 163 resulted in similar kinetic properties of the mutant form as the wt-hPAH (24-h induction) (Table III). The importance of the conserved Asn residue at this position is further supported by the finding of an Asn 167 3 Ile mutation in a PKU patient (37). Moreover, Asn 207 and Asn 223 also seem to have a structural role in hPAH.…”

supporting

confidence: 54%

Deamidations in Recombinant Human Phenylalanine Hydroxylase

Carvalho

Solstad

Bjørgo

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28°C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for Lphenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Phenylalanine hydroxylase (PAH, 1 phenylalanine 4-monooxygenase, EC 1.14.16.1) is a non-heme iron monooxygenase that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr) in the presence of the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H 4 biopterin) and dioxygen. Mutations in the human enzyme (hPAH) leading to altered kinetic properties or reduced stability of the enzyme are associated with the autosomal recessive disorder phenylketonuria (PKU). hPAH isolated from liver (2) and as recombinant enzyme expressed in Escherichia coli (3, 4) has been found to exist as a mixture of 4 -5 molecular forms with the same apparent subunit molecular mass, but with different isoelectric points (pI). Isoelectric focusing and two-dimensional electrophoresis of recombinant hPAH expressed in E. coli for 24 h at 28°C revealed five components of decreasing staining intensity and decreasing pI (denoted hPAH I-V). This microheterogeneity was shown to be the result of nonenzymatic deamidations of Asn residues (1, 4). Mainly one band with the highest pI (hPAH I) was, however, detected after a short induction period of 2 h at 28°C, and this form was considered to represent the newly synthesized and most native, nondeamidated form of the enzyme. Thus, the microheterogeneity pattern is highly dependent on the induction time with IPTG in E. coli (4). Due to the relatively high rate of deamidation, the labile amide-containing residues have been considered to be Asn residues (4), and this conclusion has recently been confirmed by the demonstration of iso-Asp in several tryptic peptides of the highly deamidated full-length wt-hPAH (1). In the deamidation reaction Asn is converted to Asp and iso-Asp in a variable ratio, sometimes with iso-Asp as the main product (5, 6). The rate of Asn deamidation in proteins and peptides has been shown to be dependent on pH, temperature, and ionic strength (7) and on intrinsic factors like the nearest neighbor amino acids, particularly the residue in the (n ϩ 1) position (8 -10). The deamidation proceeds via a cyclic succinimide intermediate (6), and with glycine in the n ϩ 1 position the rate of deamidation is unusually rapid, whereas all the other 19 amino acid residues are more sterically hindered in the formation of the cyclic intermediate (11). Moreover, the rate of deamidation is also determined by the protein secondary and tertiary structure because the necessary flexibility for the formation of the cyclic intermediate may be limited in p...

show abstract

supporting

confidence: 54%

Deamidations in Recombinant Human Phenylalanine Hydroxylase

Carvalho

Solstad

Bjørgo

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In contrast, the frequency of the most common PAH gene variations such as p.R408W, IVS12+1G>A, p.R261Q and IVS10-11G>A in Central Europe country Slovakia, (47.34%, 5.31%, 3.86% and 1.69% respectively) are similar in Slovenia [22,25]. Additionally, p.R408W is the most prevalent in Australian, American and British which accounts for 22.8%, 18.7% and 21.24% respectively [26,27,28], suggesting significant differences of distribution of PAH gene variation between Western and Eastern. The EX6-96A>G mutation was also identified in our study, with a frequency of 10.1% (15/149).…”

Section: Discussionmentioning

confidence: 91%

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China

Zhang

Gao

Feng

et al. 2018

Scandinavian Journal of Clinical and Laboratory Investigation

View full text Add to dashboard Cite

Phenylketonuria (PKU, OMIM 261600) caused by phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disease that is characterized by abnormalities of phenylalanine metabolism. In this study, a total of 77 patients, originating from the central region of China and who were diagnosed with PAH deficiency at the third affiliated hospital of Zhengzhou University, were enrolled in this study. The 13 exons and 12 flanking introns of the PAH gene were analyzed by Sanger sequencing and next generation sequencing. The sequencing data were aligned to the hg19, PAHvdb and HGMD databases to characterize the genotypes of PKU patients, and genotype-phenotype correlations and BH4 responsiveness predictions were performed using BIOPKUdb. In total, 149 alleles were characterized among the 154 PKU alleles. These mutations were located in exons 2-13, and intron 12 of the PAH gene, with a relative frequency of ≥5%, for EX6-96A>G, p.R241C, p.R243Q, p.V399V and p.R53H. Additionally, a novel variant, p.D84G, was identified. The genotype correlated with clinical symptoms in 33.3-100% of the cases, depending on the disease severity, and BH4 responsiveness predictions show that only five patients with MHP-PKU and one patient with Mild-PKU were predicted to be BH4 responsive. In conclusion, we have characterized the mutational spectrum of PAH in the central region of China and have identified a novel mutation. The hotspot mutation information might be useful for screening, diagnosis and treatment of PKU.

show abstract

Discordant phenylketonuria phenotypes in one family: the relationship between genotype and clinical outcome is a function of multiple effects.

Tyfield

Zschocke

Stephenson

et al. 1995

Journal of Medical Genetics

Self Cite

View full text Add to dashboard Cite

Four members spanning three generations of one family have phenylketonuria of varying degrees ofseverity. Two first cousins were screened in the neonatal period and have had dietary phenylalanine restriction since diagnosis, the older patient having been classified as having more severe PKU and the younger one as having mild PKU. Their mutual grandfather and his older brother also have a significant hyperphenylalaninaemia and are of normal intelligence despite never having had restricted phenylalanine intake. Mutation analysis of the phenylalanine hydroxylase (PAH) gene has established that there are four different mutations, two in exon 2 (F39L and L48S) and two in exon 3 (RlllX and S67P), which give rise to PKU in this family. In order to establish their relative severity, we screened the PKU populations of western Scotland and the south west of England for these mutations. The exon 3 mutations are rare; however, F39L is relatively common in Scotland and L48S in England. A comparison of diagnostic blood phenylalanine concentrations in subjects carrying L48S/null or F39L/null mutations with those carrying two null mutations suggest that these exon 2 mutations are less deleterious. Thus, in this family, the different biochemical phenotypes can be explained, in part, by different genotypes at the PAH locus but our results show that the relationship between genotype and clinical outcome is more complex and is a function of multiple effects.

show abstract

Mutation analysis of the phenylalanine hydroxylase gene using heteroduplex analysis with synthetic DNA constructs

Cited by 3 publications

References 6 publications

Deamidations in Recombinant Human Phenylalanine Hydroxylase

Deamidations in Recombinant Human Phenylalanine Hydroxylase

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China

Discordant phenylketonuria phenotypes in one family: the relationship between genotype and clinical outcome is a function of multiple effects.

Contact Info

Product

Resources

About