A novel missense mutation in the ALPL gene causes dysfunction of the protein

Chen,Bin; Li,Lili; Ren,Weitong; Yi,Long; Wang,Yaping; Yan,Fuhua

doi:10.3892/mmr.2017.6668

A novel missense mutation in the ALPL gene causes dysfunction of the protein

Authors:
- Bin Chen
- Lili Li
- Weitong Ren
- Long Yi
- Yaping Wang
- Fuhua Yan
View Affiliations

Affiliations: Department of Periodontology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu 210008, P.R. China, National Laboratory of Solid State Microstructure and Department of Physics, Nanjing University, Nanjing, Jiangsu 210008, P.R. China, Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China, Department of Medical Genetics, Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
Published online on: May 31, 2017 https://doi.org/10.3892/mmr.2017.6668
Pages: 710-718
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Hypophosphatasia (HP) is a rare genetic disease caused by mutation in the alkaline phosphatase, liver/bone/kidney (ALPL) gene with highly variable clinical manifestations. Efforts have been made to collect cases with novel mutations and to examine how a missense mutation affects ALPL protein function, which remains difficult to predict. The present study investigated the underlying mechanism of ALPL dysfunction in a patient diagnosed with HP. Bidirectional sequencing of the ALPL gene was conducted in a 5‑year‑old Chinese girl preliminary diagnosed with childhood HP. Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping v2 (PolyPhen‑2) tools were used to forecast the impact of the mutation on protein function. Site‑directed mutagenesis was performed and transfected into cells to verify the role of the specific mutation. Furthermore, the mechanism of the impact was investigated via all‑atom molecular dynamics (MD) simulation. The patient demonstrated a compound heterozygote with two missense mutations in the ALPL gene, p.Trp29Arg and p.Ile395Val. Trp29 and Ile395 were determined to be ‘tolerable’ by SIFT, whereas they were ‘possibly damaging’ by PolyPhen‑2 in terms of conservation. Additionally, HEK293 cells were transfected with plasmids expressing wild type and/or mutated ALPL. Only 4.1% of ALP activity remained when Trp29 was substituted by Arg, whereas 19.1, 33.7, 50.1 and 7.6% ALP activity remained in cells expressing p.Ile395Val, wild type+p.Trp29Arg, wild type+p.Ile395Val and p.Trp29Arg+p.Ile395Val substitutions, respectively. All‑atom MD simulation demonstrated that the N‑terminal helix of mutated ALPL, where Trp29 is located, separated from the main body of the protein after 30 nsec, and moved freely. These results demonstrated that p.Trp29Arg, as a novel missense mutation in the ALPL gene, reduced the enzymatic activity of ALPL. This effect may be associated with an uncontrolled N‑terminal helix. These results provide novel information about the genetic basis of HP, and may facilitate the development of future therapies.

View Figures

View References

1	Fraser D: Hypophosphatasia. Am J Med. 22:730–746. 1957. View Article : Google Scholar : PubMed/NCBI
2	Mornet E, Hofmann C, Bloch-Zupan A, Girschick H and Le Merrer M: Clinical utility gene card for: Hypophosphatasia - update 2013. Eur J Hum Genet. 22:2014.doi: 10.1038/ejhg.2013.177. View Article : Google Scholar :
3	Reibel A, Manière MC, Clauss F, Droz D, Alembik Y, Mornet E and Bloch-Zupan A: Orodental phenotype and genotype findings in all subtypes of hypophosphatasia. Orphanet J Rare Dis. 4:62009. View Article : Google Scholar : PubMed/NCBI
4	Van den Bos T, Handoko G, Niehof A, Ryan LM, Coburn SP, Whyte MP and Beertsen W: Cementum and dentin in hypophosphatasia. J Dent Res. 84:1021–1025. 2005. View Article : Google Scholar : PubMed/NCBI
5	Whyte MP: Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev. 15:439–461. 1994. View Article : Google Scholar : PubMed/NCBI
6	Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon Bouy-B, Serre JL and Mornet E: Correlations of genotype and phenotype in hypophosphatasia. Hum Mol Genet. 8:1039–1046. 1999. View Article : Google Scholar : PubMed/NCBI
7	Jemmerson R and Low MG: Phosphatidylinositol anchor of HeLa cell alkaline phosphatase. Biochemistry. 26:5703–5709. 1987. View Article : Google Scholar : PubMed/NCBI
8	Kim EE and Wyckoff HW: Structure of alkaline phosphatases. Clin Chim Acta. 186:175–187. 1990. View Article : Google Scholar : PubMed/NCBI
9	Mornet E: Hypophosphatasia. Best Pract Res Clin Rheumatol. 22:113–127. 2008. View Article : Google Scholar : PubMed/NCBI
10	Zhang W, Shen L, Deng Z, Ding Y, Mo X, Xu Z, Gao Q and Yi L: Novel missense variants of ZFPM2/FOG2 identified in conotruncal heart defect patients do not impair interaction with GATA4. PLoS One. 9:e1023792014. View Article : Google Scholar : PubMed/NCBI
11	Ho SN, Hunt HD, Horton RM, Pullen JK and Pease LR: Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 77:51–59. 1989. View Article : Google Scholar : PubMed/NCBI
12	Li W, Yang JG, Ren FX, Kang CL and Zhang SY: PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. Yi Chuan. 26:589–593. 2004.(In Chinese). PubMed/NCBI
13	Witzigmann D, Wu D, Schenk SH, Balasubramanian V, Meier W and Huwyler J: Biocompatible polymer-Peptide hybrid-based DNA nanoparticles for gene delivery. ACS Appl Mater Interfaces. 7:10446–10456. 2015. View Article : Google Scholar : PubMed/NCBI
14	Yang H, Wang L, Geng J, Yu T, Yao RE, Shen Y, Yin L, Ying D, Huang R, Zhou Y, et al: Characterization of six missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in Chinese children with hypophosphatasia. Cell Physiol Biochem. 32:635–644. 2013. View Article : Google Scholar : PubMed/NCBI
15	Le Du MH, Stigbrand T, Taussig MJ, Menez A and Stura EA: Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity. J Biol Chem. 276:9158–9165. 2001. View Article : Google Scholar : PubMed/NCBI
16	Sánchez R and Sali A: Comparative protein structure modeling. Introduction and practical examples with modeller. Methods Mol Biol. 143:97–129. 2000.PubMed/NCBI
17	Hess B, Kutzner C, van der Spoel D and Lindahl E: GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput. 4:435–447. 2008. View Article : Google Scholar : PubMed/NCBI
18	Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO and Shaw DE: Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins. 78:1950–1958. 2010.PubMed/NCBI
19	Jorgensen WL, Chandrasekhar J and Madura JD: Comparison of simple potential functions for simulating liquid water. J Chem Phys. 79:9261983. View Article : Google Scholar
20	Hess B, Bekker H, Berendsen HJC and Fraaije JG: LINCS: A linear constraint solver for molecular simulations. J Computation Chem. 18:1463–1472. 1998. View Article : Google Scholar
21	Bussi G, Donadio D and Parrinello M: Canonical sampling through velocity rescaling. J Chem Phys. 126:0141012007. View Article : Google Scholar : PubMed/NCBI
22	Parrinello M and Rahman A: Crystal Structure and Pair Potentials: A Molecular-Dynamics Study. Phys Rev Lett. 45:1196–1199. 1980. View Article : Google Scholar
23	Wenkert D, McAlister WH, Coburn SP, Zerega JA, Ryan LM, Ericson KL, Hersh JH, Mumm S and Whyte MP: Hypophosphatasia: Nonlethal disease despite skeletal presentation in utero (17 new cases and literature review). J Bone Miner Res. 26:2389–2398. 2011. View Article : Google Scholar : PubMed/NCBI
24	Silvent J, Gasse B, Mornet E and Sire JY: Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia. J Biol Chem. 289:24168–24179. 2014. View Article : Google Scholar : PubMed/NCBI
25	Hoylaerts MF, Ding L, Narisawa S, Van Kerckhoven S and Millan JL: Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment. Biochemistry. 45:9756–9766. 2006. View Article : Google Scholar : PubMed/NCBI
26	Silva I, Castelao W, Mateus M and Branco JC: Childhood hypophosphatasia with myopathy: Clinical report with recent update. Acta Reumatol Port. 37:92–96. 2012.PubMed/NCBI