Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase

Abstract

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

Phenylketonuria: Genes in phenylketonuria, diagnosis, and treatments

“…There have been identified PAH 1101 variants in PAHvdb Database; 6 about 50% are missense mutations. 7 Gu et al (2014) suggested that mutations in the PAH gene consist of 13,5% deletions, 1.8% insertions, 5% nonsense, 60.5% missense, and 11% splice site, (Pecimonova et al, 2019) . The p.Arg408Trp is the most common mutation in many populations, 2 Gundorova et al (2019) suggested that the pathogenic variant p.Arg408Trp is widespread in Europe.…”

Phenylketonuria: Genes in Phenylketonuria, Diagnosis, and Treatments

“…There have been identified PAH 1101 variants in PAHvdb Database; 6 about 50% are missense mutations. 7 Gu et al (2014) suggested that mutations in the PAH gene consist of 13,5% deletions, 1.8% insertions, 5% nonsense, 60.5% missense, and 11% splice site, (Pecimonova et al, 2019) . The p.Arg408Trp is the most common mutation in many populations, 2 Gundorova et al (2019) (Gundorova et al, 2019).…”

Characterization and expression of phenylalanine hydroxylase in rabbits under different feeding conditions of faba bean (Vicia faba L.)

“…Liver and kidney are the main organs that excrete the PAH enzyme [9,10]. The deficiency of PAH enzyme leads to the accumulation of phenylalanine (Phe) amino acid in the blood, causing phenylketonuria (PKU), a disease that causes mental retardation in infants and children [11][12][13][14][15][16][17][18][19][20][21].…”