In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU

Trunzo R, Santacroce R, Shen N, Jung-Klawitter S, Leccese A, De Girolamo G, Margaglione M, Blau N

Gene ep 9. pii: S0378-1119(16)30731-4, September 2016
DOI: 10.1016/j.gene.2016.09.015


Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

Lay summary

Hyperphenylalaninemias (HPAs) are genetic diseases mainly caused by diverse mutations in the phenylalanine hydroxylase (PAH) gene. Studies of different PAH mutations in cell cultures offer the opportunity to investigate the molecular mechanisms underlying HPAs and to clarify whether a disease-associated mutation is indeed pathogenic. Analysing the severity of a metabolic symptoms in cells carrying different mutations of the PAH gene allows determining how a mutation affects the PAH enzyme. The researchers examined eight HPAs-associated PAH mutations and found that they led to various degree of reductions of the PAH enzyme activity compared to the non-mutated enzyme. In most cases, the lower PAH activity was the more severe were the metabolic dysfunctions in the cells and the less responsive they were to tetrahydrobiopterin (BH4), used as a medicine to treat HPAs.

This study, supported by RD-Connect, has major implications for understanding of HPAs and other rare genetic diseases. They reveal how different PAH mutations result in symptoms of different severity, and why BH4 therapy is ineffective in some patients.


Further info

Click here to view the publication at the journal website