维生素D依赖性佝偻病IA型两家系的临床及分子遗传学研究

发布时间：2018-06-25 02:32

本文选题：维生素D依赖性佝偻病IA型 + CYP27B1　；参考：《福建医科大学》2015年硕士论文

【摘要】：[目的]分析2例维生素D依赖性佝偻病IA型(Vitamin D-dependent rickets type IA,VDDR-IA)患者的临床特点、个体化治疗及随访资料,以提高对本病的认识和诊治水平,并通过2个VDDR-IA患者家系CYP27B1基因突变分析,为VDDR-IA的临床诊断和治疗提供依据,同时也可丰富中国病例基因突变类型数据库。[研究对象]2例VDDR-IA患者及家系成员17人,另收集健康志愿者101人作为对照,在获得知情同意的情况下,由福建省福州儿童医院内分泌科专人采集外周血样及收集相关病历资料。所有受检人员在此前均未接受过任何遗传学相关检测。[研究方法]1.临床资料与方法1.1收集临床相关资料:详细记录2例VDDR-IA患者及其家系成员的病史,进行体格检查,并完成相关的实验室检查及影像学检查,对病历资料进行回顾性总结分析。1.2血清1,25-(OH)2D3水平测定:抽取2例患者及其家系成员外周静脉血,分离血清后应用人1,25-(OH)2D3 ELISA检测试剂盒进行血清1,25-(OH)2D3水平测定。2.CYP27B1基因测序分析2.1外周静脉血基因组DNA提取:抽取2例患者及其家系成员和101例健康对照组的外周静脉血,采用TIANGEN血液基因组DNA提取试剂盒提取基因组DNA。2.2 PCR扩增CYP27B1基因片段:参考文献资料,应用Oligo6软件进行引物验证及PCR条件分析后对CYP27B1基因9个外显子及其内含子相邻区进行PCR扩增,通过预实验确定PCR扩增最佳条件。2.3 PCR产物测序和分析:所有PCR扩增后的产物均经过1.5%琼脂糖电泳检测后,送测序公司切胶纯化后直接测序,测序结果与Blastn中CYP27B1基因参考序列(NC_000012.11与NM_000785.3)进行对比分析,发现存在的碱基变异。[结果]1.临床资料:2例患者均有严重的佝偻病症状、体征及影像学表现,实验室检查提示低钙血症、低磷血症、血清碱性磷酸酶及甲状旁腺激素水平显著升高、血清25-(OH)D3水平升高,1,25-(OH)2D3水平明显降低。两家系其他成员无相关临床表现,实验室检查结果均正常。2.CYP27B1基因测序结果:在2例VDDR-IA患者CYP27B1基因8号外显子检测到同一种纯合子突变c.1319_1325dup CCCACCC,通过对其正常表型的父系及母系成员的CYP27B1基因测序证实多人为杂合突变,101名正常对照个体CYP27B1基因相同位点测序未发现此突变。3.患者的治疗随访:予以骨化三醇和钙剂个体化治疗,2例患者症状明显改善,实验室检查提示生化指标恢复正常,影像学检查显示骨骼损害明显改善。[结论]本研究首次对来自中国同一小城市的2个完整大家系进行了基因突变分析。两例患者均为CYP27B1基因8号外显子c.1319_1325dup CCCACCC纯合突变发病,在中国VDDR-IA病例中为首次报道。治疗随访提示VDDR-IA药物应用需要个体化,对于病情严重者大剂量骨化三醇治疗是必要的,且长期应用尚未发现副作用。
[Abstract]:[objective] to analyze the clinical characteristics, individualized treatment and follow-up data of 2 patients with vitamin D-dependent rickets type IADDR-IA (Vitamin D-dependent rickets type IADDR-IA) in order to improve the understanding and diagnosis of the disease, and to analyze the CYP27B1 gene mutation in two families of VDDR-IA patients. It can provide evidence for clinical diagnosis and treatment of VDDR-IA and enrich the database of gene mutation types in Chinese cases. [participants] two VDDR-IA patients and 17 family members, and 101 healthy volunteers were collected as control. With informed consent, peripheral blood samples were collected and relevant medical records were collected by the Endocrinology Department of Fuzhou Children's Hospital in Fujian Province. None of the subjects had previously undergone any genetically-related tests. [research methods] 1. Clinical data and methods 1.1 Clinical data were collected: the history of 2 VDDR-IA patients and their family members were recorded in detail, the medical examination was performed, and the relevant laboratory and imaging examinations were completed. Retrospective analysis of medical records. 1.2 determination of serum 1O25- (OH) 2D3 levels: peripheral venous blood samples were drawn from 2 patients and their family members. After isolation of serum, Elisa kit was used to detect the level of 1m25- (OH) 2D3 in serum. 2.CYP27B1 gene sequencing analysis 2.1 peripheral venous blood genomic DNA extraction: extraction of peripheral venous blood from 2 patients and their family members and 101 healthy controls. The CYP27B1 gene fragment was amplified by TIANGEN blood genomic DNA extraction kit. 2.2 PCR. The CYP27B1 gene fragment was amplified by PCR using Oligo6 software after primer verification and PCR condition analysis, and the adjacent regions of 9 exons and introns of CYP27B1 gene were amplified by PCR. The best conditions for PCR amplification were determined by pre-experiment. 2.3 PCR products were sequenced and analyzed. All PCR products were detected by 1.5% agarose gel electrophoresis, and then sequenced directly after being purified by gumming by sequencing company. The results of sequencing were compared with the reference sequence of CYP27B1 gene in Blastn (NCSCS 000012.11 and NMSTX 000785.3). [result] 1. Clinical data: two patients had severe rickets symptoms, signs and imaging findings. Laboratory examination showed hypocalcemia, hypophosphatemia, serum alkaline phosphatase and parathyroid hormone levels increased significantly. The serum 25- (OH) D _ 3 level increased and the serum 25-(OH) _ 2D _ 3 level decreased significantly. Other members of the two families had no related clinical manifestations, Laboratory results were normal. 2. Sequencing of CYP27B1 gene: the same homozygous mutation was detected in exon 8 of CYP27B1 gene in 2 patients with VDDR-IA. The sequence of CYP27B1 gene of normal phenotype and maternal member of CYP27B1 gene was confirmed by sequencing the homozygous mutation of CYP27B1 gene. The mutation of CYP27B1 gene was not found by sequencing the same locus of CYP27B1 gene in 101 normal controls. Follow up: two patients were treated with ossification triol and calcium individualized treatment. The results of laboratory examination showed that the biochemical indexes returned to normal and the bone damage was obviously improved by imaging examination. [conclusion] for the first time, gene mutation analysis was carried out in two complete lines from the same small city in China. The homozygous mutation of exon 8 of CYP27B1 gene c.1319_1325dup CCCACCC was reported for the first time in Chinese VDDR-IA patients. The follow-up indicated that the drug use of VDDR-IA should be individualized, and it was necessary to treat severe patients with high dose of oscitic triol, and no side effects had been found in the long term use of VDDR-IA.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R591.44

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