Research paperIn vitro residual activities in 20 variants of phenylalanine hydroxylase and genotype-phenotype correlation in phenylketonuria patients
Introduction
Phenylketonuria (PKU, OMIM#261600) is a common autosomal recessive inherited metabolic disease (Blau et al., 2010; Chen et al., 2018). The prevalence of PKU varies in different areas, and in the Chinese population, it is about 1:12000 (Gu et al., 2008; Shi et al., 2012; Wang et al., 2018). The patients with phenylalanine hydroxylase (PAH) gene variants have difficulties in the conversion of phenylalanine (Phe) to tyrosine (Tyr) resulting in elevated concentrations of Phe in the blood (van Spronsen et al., 2017). The Phe concentration > 1200 μmol/L (20 mg/dL), 600–1200 μmol/L (10–20 mg/dL), and 120–600 μmol/L (2–10 mg/dL) can be considered as classic PKU (cPKU), mild PKU (mPKU) and mild hyperphenylalaninemia (MHP), respectively (Blau, 2016; Blau et al., 2010). Untreated cPKU patients are characterized by neurological disorders, behavioral disturbances, skin eczema, as well as musty odor in urine and sweat (Manta-Vogli and Schulpis, 2018; Shirzadeh and Saeidian, 2018; van Vliet et al., 2018).
As of April 12, 2019, 1111 PAH gene variants have been documented in the PAH Locus-Specific Database (PAHvdb) (http://www.biopku.org/). The residual activity of the PAH variants is the key determinant of the metabolic phenotype and BH4 responsiveness in the PKU patients (Himmelreich et al., 2018; Shen et a., 2016; Trunzo et al., 2016). Different methods have been developed for assessing PAH activity, such as colorimetric assay (Guroff et al., 1967), thin layer chromatography (TLC) (Gamez et al., 2000), high performance liquid chromatography (HPLC) coupled to fluorescence detection (Bailey and Ayling, 1980), automated fluorescence (Gersting et al., 2010), and the recently described isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Heintz et al., 2012).
Genotype-phenotype correlation is important for PKU patients. It can guide the treatment strategy and can also predict the prognosis. Different methods of genotype-based phenotype predictions have been reported, such as arbitrary values (AV) (Guldberg et al., 1998) and allelic phenotype values (APV) (Garbade et al., 2019). A study on the linking of genotype and phenotype in phenylketonuria patients demonstrated that there were significant differences in the residual activity between cPKU and mPKU/MHP, but no significant difference between mPKU and MHP has been observed (Wettstein et al., 2015). Compared to the wild-type PAH, the corresponding residual activities of mutant PAH identified in the PKU patients were 3.75 ± 4.2%, 40.4 ± 15.9% and 51.7 ± 25% for cPKU, mPKU and MHP, respectively (Wettstein et al., 2015). However, 76% of the PKU patients were found to express two compound heterozygous variants (Blau, 2016). Co-expression of two different PAH variants in vitro exhibited the phenomenon of interallelic complementation, the activity of heteromeric PAH could be higher or lower than the predicted residual activity (mean of the in vitro catalytic activities of each monoallelic variants), known as positive complementation or negative complementation, respectively (Leandro et al., 2011a, Leandro et al., 2011b; Leandro et al., 2006; Wettstein et al., 2015). Therefore, for patients with complex heterozygous variations, predicting their phenotypes is more challenging.
In this assay, we reported 9 newly discovered PAH variants, further enriched the spectrum of PAH variants. To shed further light on the correlation between PAH activity and phenotype, we expressed 20 PAH variants in COS-7 cells and detect their protein expression levels and residual activities. We also compared the results of phenotypic prediction based on APV and PAH activity respectively, and further explored the relationship between residual activity and phenotype in PKU patients.
Section snippets
Patients
Base on the clinical features, biochemical and genetic tests, a total of 1083 patients were diagnosed with PAH deficiency in the Department of Pediatric Endocrine/Genetics of Shanghai Xinhua Hospital from January 2010 to December 2016. We analyzed the spectrum of PAH gene variants and found 35 new mutations, 26 of which have been reported in our previous studies (Wang et al., 2018). We analyzed the in vitro activity in 20 of the variants (16 missense variants and 4 nonsense variants) carried by
Novel variants in PAH gene
In 7 years, 35 new PAH variants were found in 41 patients, 26 of the variants were reported in our previous article (Wang et al., 2018), 9 new variants were reported for the first time, including 2 missense variants, 1 nonsense variants, 1 splice-site variants, 3 deletion and 2 duplication. We have submitted them to PAHvdb and collected them in Fig. 1 coupled with those in our assay.
PAH protein expression and in vitro residual activities
The residual activities and protein expression of the 20 PAH variants are displayed in Table 2 and Fig. 2.
Discussion
Compared to the European countries, more people in China suffer from PKU because of the large population size. The financial burden imposed by the treatment of PKU was found to be huge because of the inadequate reimbursement policies (Wang et al., 2017). Among all the PKU patients recorded in the BIOPKU database (http://www.biopku.org), 54.8% were cPKU, 27.4% were mPKU, and 17.8% were MHP (Wettstein et al., 2015). The phenotypic distribution has large regional differences. In Eastern Europe,
Conclusion
In conclusion, we reported 9 new PAH variants for the first time, and thus, expand the spectrum of PAH variants. We also detected the in vitro residual enzyme activity of 20 PAH variants using LC-ESI-MS/MS. 12 out of 14 (86%) variants were consistently predicted by APV algorithm and residual activity, and the PAH activity accurately predicted the phenotypes in 77% of the patients. Our results contribute to the ongoing research on the genotype-phenotype correlation and BH4 responsiveness in PKU.
Details of the contributions of individual authors
Yongguo Yu, Xuefan Gu, Jun Ye, Lianshu Han, Wenjuan Qiu, Huiwen Zhang and Lili Liang collected the clinical data; Xi Mo, Qihua Fu, Nan Shen and Yue Tao designed the research; Yanjie Fan, Jianguo Wang, Zhuwen Gong and Yu Wang were responsible for genetic testing and analysis; Xia Zhang and Guoling You were responsible for the detection of enzyme activity; Xia Zhang drafted the article; Nan Shen and Xi Mo revised it critically for important intellectual content.
Funding
This study was funded by the
References (33)
- et al.
An assay for picomole levels of tyrosine and related phenols and its application to the measurement of phenylalanine hydroxylase activity
Anal. Biochem.
(1980) - et al.
The metabolic and molecular bases of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency
Mol. Genet. Metab.
(2004) - et al.
Phenylketonuria
Lancet
(2010) - et al.
The structural basis of phenylketonuria
Mol. Genet. Metab.
(1999) - et al.
Expression analysis of phenylketonuria mutations. Effect on folding and stability of the phenylalanine hydroxylase protein
J. Biol. Chem.
(2000) - et al.
Allelic phenotype values: a model for genotype-based phenotype prediction in phenylketonuria
Genet. Med.
(2019) - et al.
Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor
J. Biol. Chem.
(2010) - et al.
A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype
Am. J. Hum. Genet.
(1998) - et al.
A simple radioisotope assay for phenylalanine hydroxylase cofactor
Anal. Biochem.
(1967) - et al.
Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry
Mol. Genet. Metab.
(2012)
Relationship between genotype, phenylalanine hydroxylase expression and in vitro activity and metabolic phenotype in phenylketonuria
Mol. Genet. Metab.
Co-expression of different subunits of human phenylalanine hydroxylase: evidence of negative interallelic complementation
Biochim. Biophys. Acta
Heterotetrameric forms of human phenylalanine hydroxylase: co-expression of wild-type and mutant forms in a bicistronic system
Biochim. Biophys. Acta
Phenylketonuria as a protein misfolding disease: the mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation
Biochim. Biophys. Acta
Efficacy of sapropterin dihydrochloride (tetrahydrobiopterin, 6R-BH4) for reduction of phenylalanine concentration in patients with phenylketonuria: a phase III randomised placebo-controlled study
Lancet
Phenylketonuria dietary management and an emerging development
J. Acad. Nutr. Diet.
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These corresponding authors also contributed equally to this work.