Elsevier

Gene

Volume 707, 30 July 2019, Pages 239-245
Gene

Research paper
In vitro residual activities in 20 variants of phenylalanine hydroxylase and genotype-phenotype correlation in phenylketonuria patients

https://doi.org/10.1016/j.gene.2019.05.029Get rights and content

Highlights

  • 20 variants of Phenylalanine Hydroxylase (PAH) gene impair enzyme activity in vitro.

  • Nine PAH gene variants were reported for the first time.

  • Allelic phenotype values (APV) scores were assigned for 14 PAH variants.

  • Phenotypic predictions were consistent for 86% variants based on APV and PAH activity.

  • PAH activity correctly predicted the phenotype in 77% of phenylketonuria patients.

Abstract

Phenylketonuria (PKU), caused by phenylalanine hydroxylase (PAH) gene variants, is a common autosomal inherited metabolic disease. So far, 1111 PAH variants have been revealed. The residual activity of the PAH variants is the key determinant of the metabolic phenotype and BH4 responsiveness in PKU patients.

In this study, the spectrum of PAH variants in 1083 Chinese PKU patients was analyzed. Then 20 variants (p.L52F, p.R86P, p.L128P, p.L142P, p.D163N, p.C203G, p.E214G, p.F260L, p.M276T, p.L311R, p.P314A, p.L364F, p.Q375H, p.F382I, p.A395S, p.V412D, p.E108*, p.C203*, p.C284* and p.E353*) were expressed in COS-7 cells. The residual activities and protein expression levels were detected by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and Western blotting, respectively. We compared the results of the phenotypic prediction based on APV and PAH activity respectively, and further explored the relationship between residual activity and phenotype in PKU patients.

We reported 9 newly discovered PAH variants for the first time, thereby expanding the spectrum of PAH variants. Among the 20 variants in our assay, 8 variants showed mild impaired residual activities (48–92%) and approximately normal protein expression levels compared to the wild-type PAH. In contrast, 9 variants showed severely impaired residual activities (0–34%) and reduced protein expression. However, three variants (p.L52F, p.F260L and p.P314A) showed impaired residual activities (5%, 32% and 29%), although the proteins were well expressed. We assigned APV scores for 14 variants, in which the results of the phenotypic prediction were consistent for 12/14 (86%) variants based on APV and residual activity respectively, and the residual activity correctly predicted 17/22 (77%) of the patients. Our study helped to further understand the genotype-phenotype correlation in PKU 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

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