A 12-year-old boy with a one-year history of muscle weakness and gait disturbance that progressed slowly presented to our hospital. He had no remarkable past medical history or family history of metabolic bone disease. Roentgenographic examination revealed a deficiency of mineralization like that seen in rickets patients in the epiphysis of the bilateral proximal tibias and distal femurs ( Figure 1A ), and a radiolucent lesion with endosteal scalloping and marginal sclerosing in the left fibula ( Figure 1B ). Blood examination revealed a low serum phosphorus level of 2.3 mg/dL (reference range: 3.0-4.7 mg/dL), and a markedly high serum FGF23 level of 329 pg/mL (reference range: <50 pg/mL), and so we suspected PMT with TIO caused by the tumor-like lesion in the left fibula.
We resected the tumor en bloc by preserving the periosteum of the fibula after confirming its benignity by intraoperative frozen section diagnosis, and the cavity of the tumor was filled with beta-tricalcium phosphate (beta-TCP) ( Figure 1C  and  Supplemental Figure 1 ). The muscle weakness gradually improved, and the gait disturbance normalized within two months. In the postoperative 21-month follow-up, he had no symptoms, and hypophosphatemia was not detected. Roentgenographic examination revealed absorption of the beta-TCP and bone formation and union of the fibular shaft ( Figure 1D ).
Histopathological examination of the resected tumor revealed irregularly deposited osteoid and osteoblast-like tumor cells scattered between the osteoid and reactive osteoclastic giant cells ( Figure 1E  and  Supplemental Figure 2 ). In immunohistochemistry, expression of FGF23 was shown in the cytoplasm of the osteoblast-like tumor cells ( Figure 1F ), CD56 was diffusely positive on the cell membrane of the tumor cells and SATB2 was diffusely positive on the tumor cells ( Supplemental Figure 2 ).
After resecting the tumor, the serum FGF23 level started to decrease immediately and normalized within 3 hours ( Figure 1G ). It was also within the normal range five days after surgery. The increase in serum phosphorus level was slightly delayed as compared with that of the FGF23 level, and was observed 6 days after the operation ( Figure 1H ).
We performed RNA sequencing using a resected specimen from the patient and identified a novel in-frame fusion involving NIPBL (encoding Nipped-B gene product and fungal Scc2-type sister chromatid cohesion proteins) and BEND2 (encoding a protein which has two BEN domains in the C-terminus) ( Figure 2A ). The fusion protein contained the phosphorylation site derived from NIPBL and the BEN domain derived from BEND2 ( Figure 2B ). Whole-exome sequencing identified three point mutations (IFT172:NM_015662:exon3:c.A263G:p.N88S; VAF = 0.22, GAB2:NM_080491:exon6:c.A1409G:p.D470G; VAF = 0.15, and PRKCH : NM_006255:exon14:c.A1996T:p.I666F; VAF = 0.17), none of which
were reported as driver mutations. In polymerase chain reaction, the amplification of target regions containing breakpoint of the chromosomal structure was confirmed using two primer sets in tumor DNA of the PMT ( Supplemental Figure 3 ).
We cloned and transfected the NIPBL-BEND2 fusion gene to HEK293T and MG63 osteoblast lineage cell line. The NIPBL-BEND2 transfected cells showed faster proliferation at 48 hours after transfection (p = 0.001 and 0.003, Student's t-test, respectively). ( Figures 2C, D ). A gene set enrichment analysis of the fusion gene-introduced HEK293T cells identified a significant enrichment of MYC-target genes, consistent with faster proliferation ( Figure 2E ). However, the expression of FGF23 (log2 fold change; 0.031) and FGFR1 (log 2 fold change; 1.59) was not changed significantly by the transfection in addition to the KL/KLB, SPP1, SFRP4, and MEPE ( Supplemental Data 1 ), even though the tumor mRNA showed relatively high FPKM in these genes ( Supplemental Table 3 ).