Functional and structural characterisation of 5 missense mutations of the phenylalanine hydroxylase

Abstract

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.

Genetic Landscape and Clinical Features of Hyperphenylalaninemia in North Ossetia-Alania: High Frequency of P281L and P211T Genetic Variants in the PAH Gene

“…Eight variants exhibit high residual PAH protein activity and can be classified as mild mutations based on available data (P211T, R261Q, Q419R, I306V, A300S, E390G, V230I, A403V; Table 1). Note: n/i-not identified; *-data on the enzymatic activity of genetic variants relative to the wild type from the PAHvdb database [www.biopku.org, assessed on 6 October 2023] and from literature sources [13,[22][23][24][25][26]; **-activity not known, †-dbSNP accession number (rsID).…”

Molecular analyses of Phenylketonuria in the intellectually disabled children from Faisalabad, Punjab, Pakistan

“…According to PAH database (http://www.pahdb.mcgill.ca/), more than 567 mutations have been reported, and overall to date 950 have been identified. 6 Severity of this disorder is associated with nature and type of mutations which alter the structure and function of the enzyme. [7][8][9] Overall, the incidence of PKU is 1 out of 10,000 among Caucasians.…”