J Clin Endocrinol Metab. 2024 Aug 13;109(9):2242–2255 

PMID: 38442738 DOI: 10.1210/clinem/dgae120 

Huixiao Wu, Hui Ying, Wanyi Zhao, Yan Sun, Yanzhou Wang, Xinyu Chen, Guimei Li, Yangyang Yao, Shuo Xu, Tianyou Li, Li Fang, Xiaoqing Sun, Ning Wang, Jin Xu, Qingbo Guan, Weibo Xia, Li Wang, Ling Gao, Jiajun Zhao, Chao Xu

Highlights: This study identified a total of 21 novel genetic variants, including 16 in the PHEX gene and 5 in non-PHEX genes, within a cohort of 49 patients diagnosed with hypophosphatemic rickets (HR). Functional analyses showed that truncating variants led to the production of immature and non-glycosylated PHEX proteins, while some missense variants resulted in variable enzymatic activity. Clinical severity was found to correlate more strongly with residual PHEX enzyme activity than with mutation type alone. CRISPR/Cas9-generated mouse models supported this genotype–activity–phenotype relationship.

Background: X-linked hypophosphatemic rickets (XLHR) is a rare hereditary bone disorder caused by pathogenic variants in the PHEX gene. PHEX encodes a zinc-dependent endopeptidase involved in regulating phosphate metabolism and fibroblast growth factor 23 (FGF23). While over 640 mutations have been reported in PHEX, a clear understanding of the relationship between genotype and clinical phenotype remains elusive. This study aimed to elucidate the correlation between PHEX genotype, enzymatic activity, and disease severity.

Material and Methods: A retrospective analysis was performed on clinical and genetic data from 49 HR patients diagnosed between 2016 and 2020. Among 44 XLHR cases, 36 distinct PHEX variants were identified, 16 of which were novel. Functional characterization of selected variants included analyses of protein expression, glycosylation status, subcellular localization, and endopeptidase activity in vitro. Two novel missense variants (c.T1349C and c.C426G) were introduced into the mouse genome using CRISPR/Cas9 to generate knock-in models.

Conclusion: Truncating mutations produced immature PHEX proteins lacking proper glycosylation, confirmed via immunoblotting and immunofluorescence. Missense variants showed variable stability and enzyme activity. Residual PHEX activity showed strong positive correlation with serum phosphate levels and a composite Rickets Severity Score (RSS). In mouse models, both male and female mutants demonstrated shortened limbs, hypophosphatemia, elevated urinary phosphate excretion, increased serum ALP and FGF23 levels, cortical and trabecular bone loss, osteomalacia, and reduced mechanical bone strength. The PhexC426G mice exhibited a more severe phenotype than PhexT1349C mice, consistent with their lower enzymatic activity. 
 

This study provides compelling experimental evidence that residual PHEX enzymatic activity is a more accurate predictor of clinical severity in XLHR than mutation class alone. The newly developed mouse models offer robust tools for mechanistic studies and therapeutic development. These findings advance our understanding of the biochemical underpinnings of phosphate-wasting disorders and support activity-based approaches in personalized medicine for XLHR.

Keywords: PHEX; XLHR; genotype–PHEX activity correlation; mouse model; variant.