To explore clinical characteristics and CYP27B1 gene mutation of children with vitamin D-dependent rickets (VDDR) IA.

Two boys (case 1 and 2) with VDDR IA diagnosed in Department of Endocrinology, Children′s Hospital, Capital Institute of Pediatrics from March to December 2019 were selected as research subjects. Their clinical data of were analyzed by retrospective research methods. ①The clinical manifestations, gene detection, treatment, and follow-up results were analyzed. ②Protein function prediction softwares, such as MutationTaster, SIFT, PROVEAN, and Polyphen-2 were used to predict their pathogenicity of unreported novel mutation. Their pathogenicity of novel mutation was graded according to the ACMG Standards and Guidelines for the Interpretation of Sequence Variants developed by American College of Medical Genetics and Genomics (ACMG) in 2015. ③The conservation of novel mutation was judged by comparing protein sequences encoded by CYP27B1 gene in 8 species of primates and 100 species of vertebrates. ④Coding protein of novel mutation of CYP27B1 gene was built homologous 3D model by Rosetta software, and changes of protein structure before and after variant were analyzed. This study was approved by the Ethics Committee of Capital Institute of Pediatrics (No. SHERLL2020003).

①Age of case 1 and 2 were 5-year-7-month, and 1-year-8 month. They admitted because of " lower limb deformity for more than 3 years" and " unsteady gait since walking for 8 months" in cases collection hospital. ②They were all shorter than children of the same gender and age, and both had valgus of lower costal margin, valgus of both knees, and an " X" shape of both lower extremities. Case 1 also had caput quadratum deformity, " chicken breast" and " beaded" rib changes. ③Their serum calcium and phosphorus decreased, 25-hydroxyvitamin D3 [25(OH)-D3] slightly decreased (case 1) or normal (case 2), alkaline phosphatase (ALP) and parathyroid hormone (PTH) were significantly increased. X-ray examination of wrist and knee joints showed that there were irregular calcification zones in metaphysis of ulna and radius, and characteristic changes of rickets, such as corona radiate sign and crateriform sign. ④Case 1 carried CYP27B1 gene c. 1358G>A and c. 184C>T compound heterozygous mutation, which inherited from his mother and father, respectively, and case 2 carried c. 1325_1326insCCCACCC homozygous mutation, which inherited from his parents. Both of them were diagnosed as VDDR IA. ⑤They were treated with calcitriol and calcium, and followed up 15 months and 22 months, their symptoms and signs of rickets improved, achieving height catch-up growth, laboratory indicators returned to normal, and imaging examination showed that the bone mineral density was normal and metaphysis was smooth. ⑥Novel missense mutation c. 184C>T of CYP27B1 gene has not been reported in the Human Gene Mutation Database (HGMD) (http: //www.hgmd.cf.ac.uk). Protein functional verification suggested that this mutation was pathogenic variant. And this mutation was judged to be a probable pathogenic variant according to the ACMG Standards and Guidelines for the Interpretation of Sequence Variants. ⑦Protein sequence coded by CYP27B1 gene was highly conserved in 8 species of primates and 100 species of vertebrates. ⑧3D structure of encoded protein before and after the variant of CYP27B1 gene modeled by Rosetta software showed that the 62nd amino acid changed from histidine to tyrosine after variant, interacted with the 491nd threonine residue by one more polarity, which might cause the change of protein structure.

For children with reduction of serum calcium and phosphorus, accompanied obvious increasing of PTH and inconspicuous decreasing of 25(OH)-D3, attention should be especially paid to VDDR IA and CYP27B1 gene testing can be conducted to confirm the diagnosis. If CYP27B1 gene c. 184C>T mutation is subsequently proven to be a novel mutation by HGMD, this study will enrich the CYP27B1 gene mutation types.