Genetic analysis combined with 3D‐printing assistant surgery in diagnosis and treatment for an X‐linked hypophosphatemia patient

Abstract Background Hypophosphatemia is mainly characterized by hypophosphatemia and a low level of 1alpha,25‐Dihydroxyvitamin D2 (1,25‐(OH)2D2) and/or 1alpha,25‐Dihydroxyvitamin D3 (1,25‐(OH)2D3) in the blood. Previous studies have demonstrated that variants in PHEX and FGF23 are primarily responsible for this disease. Although patients with variants of these two genes share almost the same symptoms, they exhibit the different hereditary pattern, X‐link dominant and autosome dominant, respectively. Three‐dimensional (3D) printing is a method which can accurately reconstruct physical objects, and its applications in orthopedics can contribute to realizing a more accurate surgical performance and a better outcome. Methods An X‐linked hypophosphatemia (XLH) family was recruited, with four patients across three generations. We screened candidate genes and filtered a duplication variant in PHEX. Variant analysis and co‐segregation confirmation were then performed. Before the operation of our patient, a digital model of our patient's leg had been rebuilt upon the CT scan data, and a polylactic acid (PLA) model had been 3D‐printed. Results A novel duplication PHEX variant c.574dupG (p.A192GfsX20) was identified in a family with XLH. Its pathogenicity was confirmed by the co‐segregation assay and online bioinformatics database. The preoperative plan was made with the help of the PLA model. Then, arch osteotomy and transverse osteotomy were performed under the guidance of the previous simulation. The appearance of the surgical‐intervened leg was satisfactory. Conclusions This study identified a novel PHEX variant and showed that 3D printing tech is a very promising approach for corrective osteotomies.


| INTRODUC TI ON
Hereditary hypophosphatemia is mainly characterized by hypophosphatemia and a low level of 1alpha,25-Dihydroxyvitamin D2 (1,25-(OH) 2 D2) and/or 1alpha,25-Dihydroxyvitamin D3 (1,25-(OH) 2 D3) in the blood. This disease can be roughly divided into four main types: X-linked dominant hypophosphatemia (XLH, OMIM 307800), autosomal dominant hypophosphatemic rickets (ADHR, OMIM 193100), autosomal recessive hypophosphatemic rickets (ARHR1, OMIM 241520; ARHR2 OMIM 613312), and Xlinked recessive hypophosphatemic rickets (OMIM 300554). 1 The incidence rate of XLH is 1/20000 worldwide, and patients with XLH present various bone deformities, including genu varum and windswept lower limbs, which can be observed at a very young age. 2 XLH was first discovered as a subtype of rickets refractory to vitamin D therapy, whereas vitamin D treatment is effective in most cases of rickets. Previous studies had identified the connection between XLH and phosphate-regulating endopeptidase homolog X-linked (PHEX). 3 ADHR is another type of hereditary hypophosphatemia caused by mutated fibroblast growth factor 23 (FGF23). 1 Variants of dentin matrix acidic phosphoprotein 1 (DMP1) and ectonucleotide pyrophos- Three-dimensional (3D) printing is a method that can accurately reconstruct physical objects. It is usually applied in maxillofacial and dental surgery. However, it had been used in orthopedic surgery in recent years and achieved promising results. For example, corrective osteotomies used to be extremely complex, still, with the assistance of an accurate 3D printing model, and operations can be well performed and even virtually simulated, thus leading to a better outcome. 10 This study reported a family with four patients with XLH across three generations. Genetic analysis was performed on the proband and her parents to clarify their hereditary etiology. A novel PHEX duplication variant c.574dupG (p.A192GfsX20) was identified, which was never reported. Moreover, 3D printing was employed preoperatively, helping the surgeon to visualize the anatomy in full 3D and plan the corrective osteotomy.

| Genetic analysis
The Review Board of Xiangya Hospital of the Central South University has approved this research. Written informed consents were collected from all family members. Genomic DNA was extracted from the peripheral blood of subjects using a DNeasy Blood & Tissue Kit (Qiagen). Primer pairs of PHEX (NM_000444.4) and FGF23 (NM_020638.2) were generated, and polymerase chain reaction (PCR) was used to amplify all exons of the PHEX and FGF23 (Table S1). Direct sequencing of purified PCR products was determined using the ABI 3100 Genetic Analyzer (ABI). Sequences were analyzed using DNAMAN Software (Version 8, Lynnon Biosoft) to compare our sequencing results with the reference sequence.

| 3D-model reconstruction
To model the patient's left extremity, her leg was scanned using a 64-row helical CT machine with 0.625-mm slice thickness as previously described. 10 The scan data were output as the Digital Imaging and Communication in the Medicine standard format. CT data were read using Boholo surgical simulator software (Boholo Medical Science), and a digital 3D model was rebuilt upon this data.
The preoperative surgical plan is made, and a virtually simulated corrective osteotomy is performed on a 3D-printed polylactic acid (PLA) model.

| Case description
A XLH family with four patients across three generations was recruited in this study ( Figure 1A). The proband (III:1), a 12-year-old girl from the Central-South China, was brought to our department for the orthomorphia of lower limbs. She was first diagnosed with familial hypophosphatemic vitamin D-resistant rickets at 6 years in the local hospital, and her symptoms mainly presented gait abnormality and ever-progressive windswept lower limb deformities ( Figure 1B,C). The proband also showed a mildly elevated serum parathyroid hormone level. Her serum 25-hydroxy vitamin D concentration was less than normal (7.48 ng/mL), along with normal serum calcium and phosphate levels (Table 1). Moreover, her father (II:2) had rickets with genu varum. Two other family members had rickets (I:1 and II:3), but their blood samples were unavailable.

| Variant analysis
The known causative genes PHEX and FGF23 were examined in all available family members to elucidate the genetic mechanism of their XLH. After Sanger sequencing, a novel duplication variant of PHEX c.574dupG (p.A192GfsX20) was detected in the proband and her father ( Figure 1D). Her mother did not harbor this variant. This newly identified variant was neither found in other unaffected family members, nor reported in GnomAD (http:// gnomad.broad insti tule.org). Its pathogenicity was confirmed by the co-segregation assay and online bioinformatics database.
Adherence to the standards and guidelines of the American College of Medical Genetics and Genomics (ACMG), we classified the GATA5 variant as "Pathogenic" ( Table 2). 11 Thus, it was concluded that this variant could be the genetic etiology of XLH in this family.

| Surgical reconstruction
The preoperative examination of the proband showed normal myodynamia and muscular tension. As traction therapy might lead to stiffness of the knee, corrective surgery was performed on the patient.
The 3D printing technique was used to rebuild her deformed left femur and tibiofibular structures ex vivo as previously described. 10  After the operation, the patient's 35-degree left genu varum was repaired, and her left leg was then fixed using an Ilizarov external fixator ( Figure 1F). The appearance of the surgically intervened leg was satisfactory. The long-term effect needed further follow-up observation.

| DISCUSS ION
In this study, a novel duplication PHEX variant c.574dupG (p.A192GfsX20) was identified in a XLH family with four patients  Figure S1). Moreover, this frameshift variant of PHEX would cause a premature stop codon, which not only results in a truncated protein, but also, according to nonsense-mediated mRNA decay theory, leads to the decreased levels of PHEX mRNA in affected patients. 13 Other small insertions, such as c.466_467insAC (p.L156HfsX66) and c.682dupT (p.S228FfsX10), are located upstream or downstream from this site had been reported to be pathogenic. 14 The symptoms of the proband were consistent with previous studies in terms of PHEX loss. 15 Thus, it was reasoned that this duplication variant was the main underlying pathogenic factor in the present case. Patients with XLH can be misdiagnosed as other types of rickets with only biochemical tests because the rickets symptoms are not specific. 16 Moreover, XLH is more refractory than the standard nutritional rickets treatment. The misdiagnosed and Rocaltrol (0.5 μg/day) for patients in the family. 18 Unfortunately, the proband and her father were not provided the timely medical intervention, which resulted in severe skeletal deformities. It is challenging to confirm hypophosphatemia in infants and easily misdiagnosed as hypovitaminosis. 19

ACK N OWLED G M ENT
All subjects have consented to this study. We thank the patients and their family members for participating in this study. This work was supported by the Provincial Natural Science Foundation of Hunan (2019JJ40517) and Provincial Science and Technology Department Foundation of Hunan (2021ZK4218).

CO N FLI C T O F I NTE R E S T
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTH O R CO NTR I B UTI O N S
Jie-Yuan Jin and Li-Yang Zhang contributed to conception and design and carried out analysis and interpretation of data; Shuai Guo and Lei Zeng performed acquisition, analysis, and interpretation of data; Ke Tang carried out analysis and interpretation of data; Jie-Yu Liang contributed to conception and design and wrote the original draft; Rong Xiang revised the draft and finally approved the version.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.