Abstract
Background: X-linked hypophosphatemia (XLHR) is the most common genetic form of hypophosphatemic rickets (HR), which is caused by phosphate regulating endopeptidase homolog X-linked (PHEX) gene mutation. At present, the genotype-phenotype relationship of XLHR and the pathogenic role of PHEX are not fully understood. Methods: In this study, we summarized clinical features in a new cohort of 49 HR patients and detected 16 novel PHEX and 5 novel non-PHEX variants. Subsequently, we studied the pathogenesis of new variants by protein expression, glycosylation analysis, subcellular localization, and endopeptidase activity. Results: The results showed that missense variants (Q189H and X750R) slightly reduced protein expression without obviously altering protein length and localization, whereas truncating variants significantly impaired the synthesis of PHEX and produced a shorter immature protein in cells. Interestingly, no evident correlation was observed between mutation types and clinical phenotypes. However, when we analyzed the relationship between PHEX activity and serum phosphorus level, we found that patients with low PHEX activity tended to have severe hypophosphatemia and high rickets severity score. Following this observation, we established 2 new knock-in XLHR mouse models with 2 novel Phex variants (c.T1349C and c.C426G, respectively) using CRISPR/Cas9 technology. Both mouse models demonstrated clinical manifestations of XLHR seen in patients, and PhexC426G mice showed more severe phenotype than PhexT1349C mice, which further confirmed the rationality of genotype-PHEX enzymatic activity correlation analysis. Conclusion: Therefore, our findings demonstrated that novel PHEX variants could disrupt protein function via affecting protein synthesis, post-translational modification, cellular trafficking, and catalytic activity. Our study facilitates a better understanding of XLHR pathogenic mechanism and PHEX activity-phenotype correlation, which is of crucial importance for future diagnosis and treatment of XLHR.