A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family
Abstract Background X‐linked hypophosphatemic rickets (XLH) is a heterogeneous genetic phosphate wasting disorder that occupies the majority of inheritable hypophosphatemic rickets (HR). XLH is caused by loss‐of‐function mutations in the phosphate‐regulating endopeptidase gene (PHEX) located on the...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2020-08-01
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| Series: | Molecular Genetics & Genomic Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/mgg3.1262 |
| _version_ | 1827345668494065664 |
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| author | Baowei Li Xiong Wang Xiaodan Hao Yanran Liu Yin Wang Chan Shan Xiang Ao Ying Liu HongChu Bao Peifeng Li |
| author_facet | Baowei Li Xiong Wang Xiaodan Hao Yanran Liu Yin Wang Chan Shan Xiang Ao Ying Liu HongChu Bao Peifeng Li |
| author_sort | Baowei Li |
| collection | DOAJ |
| description | Abstract Background X‐linked hypophosphatemic rickets (XLH) is a heterogeneous genetic phosphate wasting disorder that occupies the majority of inheritable hypophosphatemic rickets (HR). XLH is caused by loss‐of‐function mutations in the phosphate‐regulating endopeptidase gene (PHEX) located on the X chromosome. Method In this study, we performed whole‐exome sequencing (WES) on the proband to identify the causative gene. The mutations were analyzed by predictive online software, such as PolyPhen‐2. Plasmids containing the wild‐type (WT) and mutant cDNA of the candidate gene were transfected into HEK293, then, the expression, cellular localization, and glycosylation state of the candidate proteins were detected by western blot, immunostaining, and endoglycosidase H digestion. The expression and concentration of related factor were measured by RT‐PCR and ELISA. Results We identified a novel missense mutation c.2179T>C in the PHEX that results in the substitution of p.Phe727Leu (F727L). This mutation was predicted to be disease‐causing by all four predictive online software. In vitro studies demonstrated that the F727L substitution hindered the intracellular trafficking of the mutant PHEX, with ~59% of mutant PHEX protein retained in the endoplasmic reticulum (ER) and only ~16% of the mutant protein localized on the cell surface. Endoglycosidase H digestion assay showed that the mutant F727L PHEX protein was not fully glycosylated. The concentration of intact FGF23 in hFOB1.19 cell culture medium collected from the mutant PHEX group was the highest (62.9 pg/ml) compared to the WT group (32.1 pg/ml) and control group (23.5 pg/ml). Conclusion Our results confirmed that the mutant PHEX protein was lowly glycosylated and retarded within the ER, the intact FGF23 level in cell culture media caused by the mutant PHEX protein was significantly elevated compared to that of the WT group, which may explain why the single base mutation in the PHEX led to XLH syndrome in this family. |
| first_indexed | 2024-03-07T23:15:53Z |
| format | Article |
| id | doaj.art-5100ab664dfc4547ac28e0f6e2cbb7e8 |
| institution | Directory Open Access Journal |
| issn | 2324-9269 |
| language | English |
| last_indexed | 2024-03-07T23:15:53Z |
| publishDate | 2020-08-01 |
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| series | Molecular Genetics & Genomic Medicine |
| spelling | doaj.art-5100ab664dfc4547ac28e0f6e2cbb7e82024-02-21T11:08:50ZengWileyMolecular Genetics & Genomic Medicine2324-92692020-08-0188n/an/a10.1002/mgg3.1262A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese familyBaowei Li0Xiong Wang1Xiaodan Hao2Yanran Liu3Yin Wang4Chan Shan5Xiang Ao6Ying Liu7HongChu Bao8Peifeng Li9Institute for Translational Medicine Qingdao University Qingdao ChinaDepartment of Reproductive Medicine Affiliated Yantai Yuhuangding Hospital of Qingdao University Yantai ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaSchool of Basic Medicine Qingdao University Qingdao ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaDepartment of Reproductive Medicine Affiliated Yantai Yuhuangding Hospital of Qingdao University Yantai ChinaInstitute for Translational Medicine Qingdao University Qingdao ChinaAbstract Background X‐linked hypophosphatemic rickets (XLH) is a heterogeneous genetic phosphate wasting disorder that occupies the majority of inheritable hypophosphatemic rickets (HR). XLH is caused by loss‐of‐function mutations in the phosphate‐regulating endopeptidase gene (PHEX) located on the X chromosome. Method In this study, we performed whole‐exome sequencing (WES) on the proband to identify the causative gene. The mutations were analyzed by predictive online software, such as PolyPhen‐2. Plasmids containing the wild‐type (WT) and mutant cDNA of the candidate gene were transfected into HEK293, then, the expression, cellular localization, and glycosylation state of the candidate proteins were detected by western blot, immunostaining, and endoglycosidase H digestion. The expression and concentration of related factor were measured by RT‐PCR and ELISA. Results We identified a novel missense mutation c.2179T>C in the PHEX that results in the substitution of p.Phe727Leu (F727L). This mutation was predicted to be disease‐causing by all four predictive online software. In vitro studies demonstrated that the F727L substitution hindered the intracellular trafficking of the mutant PHEX, with ~59% of mutant PHEX protein retained in the endoplasmic reticulum (ER) and only ~16% of the mutant protein localized on the cell surface. Endoglycosidase H digestion assay showed that the mutant F727L PHEX protein was not fully glycosylated. The concentration of intact FGF23 in hFOB1.19 cell culture medium collected from the mutant PHEX group was the highest (62.9 pg/ml) compared to the WT group (32.1 pg/ml) and control group (23.5 pg/ml). Conclusion Our results confirmed that the mutant PHEX protein was lowly glycosylated and retarded within the ER, the intact FGF23 level in cell culture media caused by the mutant PHEX protein was significantly elevated compared to that of the WT group, which may explain why the single base mutation in the PHEX led to XLH syndrome in this family.https://doi.org/10.1002/mgg3.1262glycosylationPHEXwhole‐exome sequencing (WES)X‐Linked hypophosphatemic rickets (XLH) |
| spellingShingle | Baowei Li Xiong Wang Xiaodan Hao Yanran Liu Yin Wang Chan Shan Xiang Ao Ying Liu HongChu Bao Peifeng Li A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family Molecular Genetics & Genomic Medicine glycosylation PHEX whole‐exome sequencing (WES) X‐Linked hypophosphatemic rickets (XLH) |
| title | A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family |
| title_full | A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family |
| title_fullStr | A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family |
| title_full_unstemmed | A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family |
| title_short | A novel c.2179T>C mutation blocked the intracellular transport of PHEX protein and caused X‐linked hypophosphatemic rickets in a Chinese family |
| title_sort | novel c 2179t c mutation blocked the intracellular transport of phex protein and caused x linked hypophosphatemic rickets in a chinese family |
| topic | glycosylation PHEX whole‐exome sequencing (WES) X‐Linked hypophosphatemic rickets (XLH) |
| url | https://doi.org/10.1002/mgg3.1262 |
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