探讨维生素D依赖性佝偻病(VDDR)ⅠA型患儿的临床特征，以及CYP27B1基因突变情况。

选择2019年3月至12月，于首都儿科研究所附属儿童医院内分泌科确诊为VDDR ⅠA型的2例患儿(患儿1、2)为研究对象。采用回顾性研究方法，①分析其临床表现，基因检测、治疗、转归及随访结果等。②对2例患儿检出的新突变，采用MutationTaster、SIFT、PROVEAN、Polyphen-2等蛋白质功能预测软件，预测该基因新突变的致病性。按照2015年美国医学遗传学与基因组学学会(ACMG)制定的《ACMG遗传变异分类标准与指南》，对该基因新突变致病性进行分级。③通过比对8种灵长类及100种脊椎动物CYP27B1基因编码蛋白质序列，判断新突变位点在不同物种中的保守性。④利用Rosetta软件，对CYP27B1基因新突变后编码蛋白质建立同源3D模型，分析突变前、后蛋白质结构。本研究经首都儿科研究所伦理委员会批准(审批文号：首都儿科研究所SHERLL2020003)。

①一般临床资料：患儿1、2均为男性患儿，年龄分别为5岁7个月和1岁8个月，分别因为"双下肢畸形3年"及"走路始步态不稳8个月"，于病例收集医院住院治疗。②入院查体：身高均低于同性别、年龄健康儿童，均存在肋下缘、双膝关节外翻，双下肢呈"X"型。患儿1存在方颅畸形、"鸡胸"、肋骨"串珠"样改变。③入院时实验室及影像学检查：血钙、磷均降低，25-羟维生素D3[25(OH)-D3]稍低(患儿1)或正常(患儿2)，碱性磷酸酶(ALP)、甲状旁腺素(PTH)均明显升高。腕、膝关节X射线摄片检查均可见尺、桡骨干骺端先期钙化带不规则，放射冠征、杯口征等佝偻病特征性改变。④基因检测：患儿1存在CYP27B1基因c.1358G>A与c.184C>T复合杂合突变；患儿2存在c.1325_1326insCCCACCC纯合突变，均遗传自其父母，被确诊为VDDR ⅠA型。⑤治疗及随访：对2例患儿采取骨化三醇及钙剂口服治疗后分别随访15个月和22个月，佝偻病相应症状、体征好转，出现追赶生长，实验室及影像学指标恢复正常，可见干骺端光整。⑥新突变致病性及其分级：CYP27B1基因c.184C>T在人类基因突变数据库(HGMD)(http：//www.hgmd.cf.ac.uk)中尚未收载，为新突变。采用MutationTaster、SIFT、PROVEAN、Polyphen-2蛋白质功能预测软件分析发现，该新突变具有致病性；根据《ACMG遗传变异分类标准与指南》，该新突变被判断为可能致病性突变。⑦新突变位点在不同物种中的保守性分析：对CYP27B1基因该新突变位点编码蛋白质序列，在8种灵长类及100种脊椎动物中的保守性分析发现，该位点高度保守。⑧新突变前、后编码蛋白质三维结构分析：采用Rosetta软件对CYP27B1基因c.184C>T突变前、后编码蛋白质同源3D模型发现，该位点突变后，第62位氨基酸由组氨酸突变成酪氨酸后，增加了与第491位苏氨酸残基的极性相互作用，可能引起编码蛋白质结构变化。

对于血钙、磷均降低，伴有PTH升高，25(OH)-D3非明显降低患儿，需考虑VDDR ⅠA型可能，应采取CYP27B1基因检测确诊。如果CYP27B1基因c.184C>T突变后续被HGMD证明是新突变，则本研究将丰富CYP27B1基因突变类型。

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.