In vitro expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase

PANG Yong-Hong; GAO Xiang-Yu; YUAN Zhen-Ya; HUANG Hui; WANG Zeng-Qin; PENG Lei; LI Yi-Qun; LIU Jie; LIU Dong; CHEN Gui-Rong

doi:10.7499/j.issn.1008-8830.2309035

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Chinese Journal of Contemporary Pediatrics ›› 2024, Vol. 26 ›› Issue (2) : 188-193. DOI: 10.7499/j.issn.1008-8830.2309035

EXPERIMENTAL RESEARCH

In vitro expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase

PANG Yong-Hong, GAO Xiang-Yu, YUAN Zhen-Ya, HUANG Hui, WANG Zeng-Qin, PENG Lei, LI Yi-Qun, LIU Jie, LIU Dong, CHEN Gui-Rong

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Abstract

Objective To study the in vitro expression of three phenylalanine hydroxylase (PAH) mutants (p.R243Q, p.R241C, and p.Y356X) and determine their pathogenicity. Methods Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein. Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells. Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants, and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay. Results Bioinformatics analysis predicted that all three mutants were pathogenic. The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control (P>0.05), while the mRNA expression level of the p.Y356X mutant significantly decreased (P<0.05). The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control (P<0.05). The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control (P<0.05), while there was no significant difference between the p.R243Q mutant and the wild type control (P>0.05). Conclusions p.R243Q, p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells, with p.R241C and p.Y356X mutants also affecting the function of PAH protein. These three PAH mutants are to be pathogenic.

Key words

Phenylalanine hydroxylase deficiency / Mutant / In vitro expression / Cell

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PANG Yong-Hong, GAO Xiang-Yu, YUAN Zhen-Ya, HUANG Hui, WANG Zeng-Qin, PENG Lei, LI Yi-Qun, LIU Jie, LIU Dong, CHEN Gui-Rong. In vitro expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase[J]. Chinese Journal of Contemporary Pediatrics. 2024, 26(2): 188-193 https://doi.org/10.7499/j.issn.1008-8830.2309035

References

1 Foreman PK, Margulis AV, Alexander K, et al. Birth prevalence of phenylalanine hydroxylase deficiency: a systematic literature review and meta-analysis[J]. Orphanet J Rare Dis, 2021, 16(1): 253. PMID: 34082800. PMCID: PMC8173927. DOI: 10.1186/s13023-021-01874-6.
2 Zhan JY, Qin YF, Zhao ZY. Neonatal screening for congenital hypothyroidism and phenylketonuria in China[J]. World J Pediatr, 2009, 5(2): 136-139. PMID: 19718537. DOI: 10.1007/s12519-009-0027-0.
3 van Wegberg AMJ, MacDonald A, Ahring K, et al. The complete European guidelines on phenylketonuria: diagnosis and treatment[J]. Orphanet J Rare Dis, 2017, 12(1): 162. PMID: 29025426. PMCID: PMC5639803. DOI: 10.1186/s13023-017-0685-2.
4 Blau N. Genetics of phenylketonuria: then and now[J]. Hum Mutat, 2016, 37(6): 508-515. PMID: 26919687. DOI: 10.1002/humu.22980.
5 Opladen T, López-Laso E, Cortès-Saladelafont E, et al. Consensus guideline for the diagnosis and treatment of tetrahydrobiopterin (BH4) deficiencies[J]. Orphanet J Rare Dis, 2020, 15(1): 126. PMID: 32456656. PMCID: PMC7251883. DOI: 10.1186/s13023-020-01379-8.
6 Jervis GA. Phenylpyruvic oligophrenia deficiency of phenylalanine-oxidizing system[J]. Proc Soc Exp Biol Med, 1953, 82(3): 514-515. PMID: 13047448.
7 van Spronsen FJ, van Wegberg AM, Ahring K, et al. Key European guidelines for the diagnosis and management of patients with phenylketonuria[J]. Lancet Diabetes Endocrinol, 2017, 5(9): 743-756. PMID: 28082082. DOI: 10.1016/S2213-8587(16)30320-5.
8 Huijbregts SCJ, Bosch AM, Simons QA, et al. The impact of metabolic control and tetrahydrobiopterin treatment on health related quality of life of patients with early-treated phenylketonuria: a PKU-COBESO study[J]. Mol Genet Metab, 2018, 125(1/2): 96-103. PMID: 30007854. DOI: 10.1016/j.ymgme.2018.07.002.
9 Vieira E Neto, Maia HS Filho , Monteiro CB, et al. Quality of life and adherence to treatment in early-treated Brazilian phenylketonuria pediatric patients[J]. Braz J Med Biol Res, 2017, 51(2): e6709. PMID: 29267500. PMCID: PMC5731329. DOI: 10.1590/1414-431X20176709.
10 Crujeiras V, Aldámiz-Echevarría L, Dalmau J, et al. Vitamin and mineral status in patients with hyperphenylalaninemia[J]. Mol Genet Metab, 2015, 115(4): 145-150. PMID: 26123187. DOI: 10.1016/j.ymgme.2015.06.010.
11 Kose E, Arslan N. Vitamin/mineral and micronutrient status in patients with classical phenylketonuria[J]. Clin Nutr, 2019, 38(1): 197-203. PMID: 29433755. DOI: 10.1016/j.clnu.2018.01.034.
12 Burlina AP, Lachmann RH, Manara R, et al. The neurological and psychological phenotype of adult patients with early-treated phenylketonuria: a systematic review[J]. J Inherit Metab Dis, 2019, 42(2): 209-219. PMID: 30690773. DOI: 10.1002/jimd.12065.
13 Klimek A, Baerwald C, Schwarz M, et al. Everyday life, dietary practices, and health conditions of adult PKU patients: a multicenter, cross-sectional study[J]. Ann Nutr Metab, 2020, 76(4): 251-258. PMID: 32998147. DOI: 10.1159/000510260.
14 Trefz F, Muntau AC, Schneider KM, et al. Health economic burden of patients with phenylketonuria (PKU): a retrospective study of German health insurance claims data[J]. Mol Genet Metab Rep, 2021, 27: 100764. PMID: 34036045. PMCID: PMC8138676. DOI: 10.1016/j.ymgmr.2021.100764.
15 庞永红, 褚英, 刘雪楠, 等. 淮海地区汉族苯丙酮尿症患儿苯丙氨酸羟化酶基因突变分析[J]. 中华实用儿科临床杂志, 2018, 33(20): 1545-1549. DOI:10.3760/cma.j.issn.2095-428X.2018.20.006.
16 王秋菊, 沈亦平, 邬玲仟, 等. 遗传变异分类标准与指南[J]. 中国科学（生命科学）, 2017, 47(6): 668-688. DOI: 10.1360/N052017-00099.
17 Liu N, Huang Q, Li Q, et al. Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China[J]. BMC Med Genet, 2017, 18(1): 108. PMID: 28982351. PMCID: PMC5629770. DOI: 10.1186/s12881-017-0467-7.
18 Hillert A, Anikster Y, Belanger-Quintana A, et al. The genetic landscape and epidemiology of phenylketonuria[J]. Am J Hum Genet, 2020, 107(2): 234-250. PMID: 32668217. PMCID: PMC7413859. DOI: 10.1016/j.ajhg.2020.06.006.
19 Mitchell JJ, Trakadis YJ, Scriver CR. Phenylalanine hydroxylase deficiency[J]. Genet Med, 2011, 13(8): 697-707. PMID: 21555948. DOI: 10.1097/GIM.0b013e3182141b48.
20 Zong Y, Liu N, Ma S, et al. Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro[J]. Gene, 2018, 668: 135-139. PMID: 29653233. DOI: 10.1016/j.gene.2018.03.078.
21 Garbade SF, Shen N, Himmelreich N, et al. Allelic phenotype values: a model for genotype-based phenotype prediction in phenylketonuria[J]. Genet Med, 2019, 21(3): 580-590. PMID: 29997390. DOI: 10.1038/s41436-018-0081-x.
22 Pey AL, Desviat LR, Gámez A, et al. Phenylketonuria: genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH[J]. Hum Mutat, 2003, 21(4): 370-378. PMID: 12655546. DOI: 10.1002/humu.10198.
23 Lee DH, Koo SK, Lee KS, et al. The molecular basis of phenylketonuria in Koreans[J]. J Hum Genet, 2004, 49(11): 617-621. PMID: 15503242. DOI: 10.1007/s10038-004-0197-5.
24 Himmelreich N, Shen N, Okun JG, et al. Relationship between genotype, phenylalanine hydroxylase expression and in vitro activity and metabolic phenotype in phenylketonuria[J]. Mol Genet Metab, 2018, 125(1/2): 86-95. PMID: 30037505. DOI: 10.1016/j.ymgme.2018.06.011.
25 Sarodaya N, Tyagi A, Kim HJ, et al. Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants[J]. Sci Rep, 2022, 12(1): 14243. PMID: 35987969. PMCID: PMC9392723. DOI: 10.1038/s41598-022-18656-0.
26 Waters PJ. How PAH gene mutations cause hyper-phenylalaninemia and why mechanism matters: insights from in vitro expression[J]. Hum Mutat, 2003, 21(4): 357-369. PMID: 12655545. DOI: 10.1002/humu.10197.
27 Kaufman RJ. Vectors used for expression in mammalian cells[J]. Methods Enzymol, 1990, 185: 487-511. PMID: 2381327. DOI: 10.1016/0076-6879(90)85041-l.
28 van Spronsen FJ, Blau N, Harding C, et al. Phenylketonuria[J]. Nat Rev Dis Primers, 2021, 7(1): 36. PMID: 34017006. PMCID: PMC8591558. DOI: 10.1038/s41572-021-00267-0.
29 Shen N, Heintz C, Thiel C, et al. Co-expression of phenylalanine hydroxylase variants and effects of interallelic complementation on in vitro enzyme activity and genotype-phenotype correlation[J]. Mol Genet Metab, 2016, 117(3): 328-335. PMID: 26803807. DOI: 10.1016/j.ymgme.2016.01.004.
30 Blau N, Martinez A, Hoffmann GF, et al. DNAJC12 deficiency: a new strategy in the diagnosis of hyperphenylalaninemias[J]. Mol Genet Metab, 2018, 123(1): 1-5. PMID: 29174366. DOI: 10.1016/j.ymgme.2017.11.005.