Six ALPL gene variants in five children with hypophosphatasia

Na Su; Min Zhu; Xinran Cheng; Ke Xu; Roland Kocijan; Huijiao Zhang

doi:10.21037/atm-21-2096

Six ALPL gene variants in five children with hypophosphatasia

Ann Transl Med. 2021 May;9(10):888. doi: 10.21037/atm-21-2096.

Authors

Na Su^{1

2}, Min Zhu¹, Xinran Cheng², Ke Xu², Roland Kocijan^{3

4}, Huijiao Zhang¹

Affiliations

¹ Department of Endocrinology, Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing, China.
² Department of Child Endocrinology and Genetic Metabolism, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
³ Medical Faculty of Bone Diseases, Sigmund Freud University, Vienna, Austria.
⁴ Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of OEGK and AUVA Trauma Center Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria.

Abstract

Background: Hypophosphatasia (HPP) is a rare hereditary disorder characterized by defective bone and tooth mineralization caused by mutations in the alkaline phosphatase (ALPL) gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). Here we performed clinical and molecular studies on 5 HPP children to investigate the pathogenic mechanisms of the ALPL gene variants.

Methods: Clinical and genetic analyses were performed on 5 HPP children, and the loci where ALPL variants were identified. Plasmids containing the relevant loci were constructed. The molecular and cellular mechanisms of the pathogenic ALPL variants were investigated by cellular immunofluorescence, enzyme activity assay, and protein expression assay.

Results: A total of 6 ALPL variants were identified in 5 HPP children: proband 1: c.346G>A (p.A116T); proband 2: c.346G>A (p.A116T)/deletions from c.1097 to c.1099 CCT (p.T366_S367deli) compound heterozygous variant; proband 3: insertion of G from c.1014 to c.1015 (p.H338fs)/c.1446C>A (p.H482Q) compound heterozygous variant; proband 4: c.920C>T (p.P307L); and proband 5: c.883A>G (p.M295V). Twenty-four hours after the HEK-293T was transfected with different variant plasmids, its alkaline phosphatase activity and enzyme protein content were reduced compared with the wild type, and there were differences among different variants. Except for 1014-G-1015+C1446A, the degree of reduction in enzyme activity was negatively correlated with the severity of clinical manifestations. Immunofluorescence revealed that the variants (especially c.883A>G and c.920C>T) caused a decrease in alkaline phosphatase expression in the cellular membrane.

Conclusions: In total, 3 novel variants were identified in these 5 HPP children, the discovery of which will enrich the human ALPL gene mutation database. Different variants in the ALPL gene can downregulate the activity of TNSALP enzyme (and thus affect its function) by affecting protein expression and translational modifications. The same variant may cause clinical manifestations of different severities in different individuals due to the presence of dominant negative effects, alterations in noncoding sequences, blind area of intron regulatory region sequencing, and variations in environmental and individual factors. The molecular mechanisms via which the ALPL gene exerts its expression effect in vivo are highly variable and warrant further investigation.

Keywords: Hypophosphatasia; alkaline phosphatase; gene variant; pathogenic mechanism; the liver/bone/kidney alkaline phosphatase gene (ALPL gene).