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.

Keywords: BH4; functional studies; missense variants; phenylalanine hydroxylase; phenylketonuria.

Figure 1

Top view (left) and side view (right) on the X-ray tetrameric rat PAH structure with color monomers. Gly239 is highlighted in sphere representation in red, Ala342 is in green, and Fe ion in the active site is in cyan.

Figure 2

Mutant PAH protein levels compared to WT PAH expressed in HepG2 cells in the presence and absence of BH-4 precursor sepiapterin. The results were calculated as a mean of three independent Western blot analyses performed from three independent PAH expressions in HepG2 cells. *** statistically significant level changes with the p-value < 0.0001 between mutated proteins expressed in the presence and absence of sepiapterin.