XLH is the most prevalent type of hereditary hypophosphatemic rickets. It is characterized by an X-linked dominant inheritance and has an incidence rate of approximately 3.9 to 5 per 100,000 individuals (1). XLH patients commonly exhibit clinical manifestations such as progressive skeletal deformities, growth retardation, and dental hypoplasia. However, the severity of these manifestations varies greatly, particularly in terms of femoral morphology (2). Joint pain and osteoarthritis are frequent complications experienced by XLH patients due to skeletal deformities, fractures, enthesopathy, spinal stenosis, and hearing loss (3).

In 1995, an international collaborative research team identified XLH as a genetic disorder caused by loss-of-function mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) (4). The PHEX gene (NM_000444.6), located in the Xp22.2-p.22.1 region of the X chromosome, contains the full genome sequence of 219 kb and comprises 22 exons. This gene encodes a transmembrane protein with 749 amino acids, belonging to the type II integral membrane zinc-dependent endopeptidase family. Mutations in the PHEX gene can cause elevated levels of circulating fibroblast growth factor 23 (FGF23), but the exact mechanism is not fully understood (5).

As XLH is an X-linked dominant disorder, the manifestation of this disorder requires only one mutant allele. While the presence of a normal allele may mitigate the severity of XLH-related bone defects, the clinical phenotypes can still vary among affected individuals within the same family. Several studies have demonstrated that adult male patients tend to exhibit more severe skeletal phenotypes compared to heterozygous female patients, including lower limb deformities, short stature, and dental abnormalities (6, 7). However, there is ongoing debate regarding whether these differences are attributed to the presence of one normal allele in heterozygous female patients (6, 8–14).

In this study, we conducted an analysis of growth and development, serum biochemical parameters, expression of phosphorus regulators, and skeletal phenotypes in order to investigate the relationship between the dosage of PHEX mutation and clinical phenotypes using a rat model. The objective of this research is to enhance the quality of life for families with children affected by XLH through early intervention measures.

Two cases of rickets from the same family, a mother and her child, were enrolled in this study ( Figure 1A ). The proband boy (III-2) born to a non-consanguineous couple. Both patients exhibited common clinical manifestations, including short stature, genu varus deformity, and tooth hypoplasia. The adult female patient, aged 36 and measuring 148 cm in height, reported a history of rickets during her childhood, characterized by genu varus deformity in both lower extremities and musculoskeletal pain symptoms. The male child patient, aged 8 and measuring 115 cm in height, developed genu varus deformity in both lower extremities at the age of 2, accompanied by cephalus quadratus and pigeon breast. Blood tests (serology) were performed on both patients, revealing decreased levels of phosphorus, normal levels of calcium and 25-(OH)₂D₃, as well as significantly elevated levels of parathyroid hormone (PTH) and alkaline phosphatase (ALP) ( Table 1 ).

Venous blood samples were collected from two patients, and DNA samples were extracted using column chromatography. The extraction process was conducted by KingMed (Guangzhou, China) for subsequent whole-exome gene sequencing analysis. Through this analysis, an A-base deletion at position 1985 (c.1985delA) was identified in the PHEX-encoding gene ( Figure 1B ). Interestingly, the c.1985del (p.Asn662fs) variation of the PHEX gene has been found in ClinVar, indicating its association with disease. However, research specifically investigating this genetic mutation is currently lacking. In order to determine its pathogenicity, an assessment of the gene was conducted following the guidelines provided by the American College of Medical Genetics and Genomics (ACMG). Inheritance models and published evidence were also taken into consideration. Based on the assessment, it has been determined that the c.1985del (p.Asn662fs) variation of the PHEX gene is a pathogenic mutation. This determination is supported by several criteria: PVS1-the patients had null variant in a gene where loss of function is a known mechanism of disease; PM2-the variant was absent from controls in gene mutation databases; PP1-the variant showed cosegregation with the disease in multiple affected family members in a gene definitively known to cause the disease; PP4-the patient’s phenotype or family history was highly specific for a disease with a single genetic etiology.

Overall, these findings indicate that the c.1985del (p.Asn662fs) variation of the PHEX gene is a pathogenic mutation associated with the development of low-phosphate rickets. Further research is warranted to explore its functional implications and potential therapeutic targets.

This deletion leads to frameshifts, resulting in premature termination of translation and the generation of a truncated protein called p.Asn662Metfs*25. In order to investigate the underlying structural mechanism of protein dysfunction caused by the frameshift mutation (Asn662fs), we employed AlphaFold to predict the structures of both the wild-type and mutant proteins. Our analysis revealed that the frameshift mutation leads to the deletion of the C-terminal segment (663-749) within the peptidase domain. This missing segment (MS) is located in close proximity to the active site and metal ion binding sites. Moreover, it was observed that Arg692 and Arg716 within the MS region form hydrogen bonds with the proton donor site (Asp646) ( Figure 2 ). Based on these findings, we speculate that the frameshift mutation disrupts the functional activity of the PHEX protein ( Supplementary Figure 2 ).

In this study, we developed a rat model to investigate the association between deletion mutations in the PHEX gene and XLH. By utilizing this model, we compared the phenotypes of rats with five different genotypes to examine the correlation between PHEX gene mutation dosage and clinical manifestations.

XLH is a metabolic bone disease that is inherited in an X-linked dominant manner. It is primarily caused by the loss of function or mutation of the PHEX gene. This genetic abnormality leads to impaired reabsorption of phosphate in the renal tubules, along with increased phosphate excretion. Consequently, affected individuals exhibit impaired mineralization of the epiphysis and cartilage. In this study, we conducted an investigation on two patients diagnosed with rickets and identified a deletion mutation (c.1985delA) in the PHEX gene through whole-exome gene sequencing analysis. This deletion mutation caused disruption in the functional activity of the PHEX protein. Subsequently, we utilized CRISPR/Cas9-mediated gene editing to successfully generate a rat model of low-phosphate rickets with the same PHEX gene mutation (c.1985delA). The purpose of this model was to comprehensively investigate the impact of this frameshift mutation on the development and progression of XLH. It is essential to note that this rat model differs from XLH rat models, such as Hyp and Ska1. Through phenotype analysis, we observed several distinct characteristics in the rat model, including delayed growth and development, a significant decrease in blood phosphorus concentration, an evident increase in FGF23 expression, as well as stubby femurs, femur deformities, and reduced BMD. These observed phenotypes were consistent with the clinical presentations of patients with XLH and previous findings from animal models (15–18).

XLH is inherited in an X-linked dominant manner, with females having two X chromosomes. If either X chromosome carries the pathogenic gene, the disease will occur. Previous studies have indicated that the PHEX gene is regulated by a dosage compensation mechanism involving random inactivation of one X chromosome (19). However, the relationship between the severity of clinical phenotypes in XLH patients and the gene dosage associated with the X-linked dominant inheritance pattern has been a subject of intense debate (6–8, 12, 13). Regarding serology, previous research has demonstrated that differences in gene dosage between male and female XLH patients have no impact on biochemical test results (7, 12), even in the first patient known to have a homozygous PHEX mutation (20). In our study, we observed that homozygous females with a double PHEX gene mutation had the lowest phosphorus levels and the highest concentration of FGF23. A similar pattern was observed for ALP levels. On the other hand, heterozygous females with one normal allele showed a slight improvement in some serum biochemical parameters, such as phosphorus, ALP, and FGF23, but these effects were not statistically significant. It is possible that the PHEX mutation gene has reduced the set point of extracellular phosphorus concentration, thereby maintaining phosphorus and FGF23 levels at the desired range. Based on these findings, we propose the hypothesis that the dosage of PHEX gene mutation may have an impact on ALP, phosphorus, and FGF23 concentrations.

Studies have indicated that a new mouse model for XLH disease, induced by N-ethyl-N-nitrosourea (ENU) and carrying the PHEX ^L222P mutation, exhibits an overexpression of PHEX transcript and protein, resulting in a loss-of-function phenotype (21). However, our findings in the mutant (c.1985delA) rat model revealed reduced expression of PHEX mRNA. We propose that this mutation leads to immature mRNA expression, which is vulnerable to degradation, consequently resulting in decreased expression levels. Regarding phosphorus regulators and metabolic factors, FGF23, released from osteoblasts and osteoclasts, acts on the kidney (22) by binding to the Klotho (Kl):FGF receptor complex (23, 24). This complex inhibits renal phosphate reabsorption and enhances phosphate excretion in the renal tubules by reducing the expression of sodium-driven phosphate cotransporters NaPi-IIa (Slc34a1) and NaPi-IIc (Slc34a3) (25–27). Phosphate regulators, such as MEPE and SFRP-4, play crucial roles in maintaining phosphate homeostasis (28, 29). Overexpression of MEPE and SFRP-4 can lead to abnormal renal phosphate metabolism and bone mineralization, causing hypophosphatemia and increased renal phosphate excretion (30, 31). Previous studies have suggested a potential role of MEPE in the pathogenesis of XLH (32), as elevated levels of MEPE were observed in XLH patients (33). In our study, we found no significant differences in the levels of FGF23, MEPE, SFRP-4, Kl, Slc34a1, and Slc34a3 among the mutant rats. Furthermore, there was no apparent dosage effect, consistent with previous relevant studies (16). Based on our data, it appears that the dosage of the PHEX mutation does not significantly influence phosphate homeostasis in bone and kidney tissues.

It was revealed that the presence of a normal PHEX allele greatly enhanced bone mineral density levels in female rat with heterozygous mutations (16). In comparison to female patients with heterozygous mutations, male patients with XLH may exhibit more severe bone and dental characteristics (6–8, 13). The presence of two mutant alleles had a significant impact on bone histomorphometry, with homozygous females displaying shorter vertebral body lengths compared to heterozygous females (34). In this study, we observed that double PHEX gene mutation in twelve-week-old mutant rats resulted in significantly shorter and smaller femurs. This indicates that the dosage of PHEX gene mutation negatively affects bone metabolism and long bone growth. Interestingly, the presence of one normal allele was found to improve the abnormal long bone growth. Further analysis using µCT revealed that PHEX gene mutation led to reduced bone mass and trabeculae in cancellous bone, as well as significant thinning of the cortical bone. However, the presence of one normal allele did not have a significant impact on the bone parameters of cancellous and cortical bones.

Our findings suggest that while the extent of abnormal mineral metabolism remains largely unchanged, the presence of one normal allele partially improves the bone’s abnormal mineralization and osteopenia, reducing the severity of intrinsic bone defects in individuals with XLH. Nevertheless, the differences in skeletal parameters are not substantial. Based on these observations, we propose that PHEX may have a role in bone mineralization that is independent of phosphate recognition. Alternatively, cells expressing one normal PHEX allele might partially rescue the defects present in those lacking PHEX.

A new XLH rat model was successfully developed by CRISPR/Cas9-mediated gene editing, which indicated that the deletion mutation of the PHEX gene locus could lead to the initiation of XLH and the model exhibited obvious clinical manifestations of XLH.

Throughout the growth period of female mutant rats, significant the dosage effects were identified in terms of weight gain, body size, tail shape, femur length and morphology, as well as the metabolism of ALP, phosphorus, and FGF23. The presence of one normal allele partially attenuated the severity of the clinical phenotype in XLH. These results highlight the possibility that PHEX gene mutations may directly impair bone metabolism through an as-yet-unknown mechanism.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was approved by the lab of animal experimental ethical inspection. All animal study protocols was approved by the ethics committee of Affiliated Hospital of Guangdong Medical University (approval number: AHGDMU-LAC-II (1)-2211-B063). The study was conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

XC and CC contributed to the conception and design of this study, the performance of experiments, interpretation, data analysis and manuscript writing. SL, FS, CM, JL and WP contributed to the performance of experiments and data analysis. QY, YC, YW and TF performed data analysis and interpretation. XC and GM contributed to the design of this study and acquiring financial support. ML and GM contributed to the data analysis, interpretation and the final approval of the manuscript. All authors contributed to the article and approved the submitted version.