【作者】 覃磊；

【导师】 邬玲仟； 梁德生； 潘乾； 龙志高；

【作者基本信息】 中南大学 ， 遗传学， 2010， 硕士

【摘要】 背景：锁骨颅骨发育不全(Cleidocranial Dysplasia,CCD)是一种罕见的遗传性骨骼系统疾病(MIM 119600),出生发病率为1：100,000。该综合征多为常染色体显性遗传方式,其致病基因定位于染色体6p21。以往研究表明成骨细胞特异性转录因子(RUNX2/CBFA1)的杂合突变、基因插入、缺失等是造成CCD的重要原因。此外,该病具有极强的外显率和明显的家族聚集性,且性别间无明显差异。典型的CCD临床表现包括：锁骨发育不良或无锁骨,囟门和颅缝增宽、延迟闭合或不闭合,身材矮小,出牙晚和恒牙数目增多等骨骼异常。这些骨骼异常所造成的身体畸形给患者带来了沉重的生活压力和心理负担。目的：通过对收集到的两个锁骨颅骨发育不全家系的RUNX2基因突变研究,进一步探索该病的分子发病机制。方法：对临床收集到的两个锁骨颅骨发育不全综合征家系行外周血基因组DNA的提取,利用聚合酶链式反应,DNA直接测序检测突变。对检测到的突变用DHPLC方法在健康人群中进行筛查。对未发现RUNX2基因位点突变的患者行荧光原位杂交检测及全基因组拷贝数检测。结果：家系一中每位CCD患者均存在RUNX2基因c.507delC的杂合突变,从而使171位氨基酸的密码子CTG移码变成TGA而提前终止,改变了RUNX2基因中runt功能域的氨基酸序列。家系二中患者DNA测序未发现RUNX2基因突变,后经包含RUNX2基因的BAC探针荧光原位杂交以及全基因组拷贝数分析显示该患者存在包含RUNX2基因在内的3.5Mb大片段杂合缺失。结论：本研究在家系一患者中检测到的杂合点突变c.507delC造成RUNX2基因编码氨基酸发生移码改变而提前终止。在家系二患者中检测到的3.5Mb大片段杂合缺失包含了整个RUNX2基因,使得RUNX2编码蛋白质的功能完全缺乏。这两种基因组改变分别是引起两家系中CCD患者患病的原因。这两种突变类型是在锁骨颅骨发育不全患者中首次被检测到的RUNX2基因新突变。两种新突变类型的确立进一步扩展了CCD的突变谱,将会对CCD产前诊断和植入前诊断产生积极的影响。更多还原

【Abstract】 Backgrounds:Cleidocranial dysplasia (CCD, MIM 119600) is a rare hereditary skeletal disorder. Its disease rate at birth is about 1:100 000. CCD is a dominantly inherited disease and the locus has been mapped to chromosome 6q21. Previous study shows that heterozygous mutations including insertions and deletions are the main cause of CCD. Besides, CCD shows strong penetrance and apparente familial aggregation. There is no obvious difference between male and female patients. CCD is characterized by patent fontanelles, wide cranial sutures, hypoplasia of clavicles, short stature, supernumerary teeth, and other skeletal anomalies. These skeletal anomalies may cause both mentally and physically prodigious pains.Objective:To further explore the molecular pathogenesis of cleidocranial dysplasia by gene mutation studying of the two collected CCD pedigrees.Methods:A sporadic patient and a four-generation family with the clinical diagnosis of cleidocranial dysplasia was investigated in this study. Genomic DNA was extracted from peripheral blood samples of each selected family member. Direct sequencing of the PCR products of the coding region of RUNX2 gene was used to identify the mutations. We used denaturing high-performance liquid chromatography (DHPLC) to exclude the possibility of happening of the sequenced mutation in unrelated normal individuals. And then detections of fluorescence in situ hybridization (FISH) and whole genome copy number analysis were applied on those patients whose sequenced region was wild type.Results:In each patient of the four-generation pedigree 1, a de novo heterozygous point mutation, cDNA 507 del C, was detected in RUNX2 exon 2. And a 3.5Mb large heterozygous deletion including the whole region of RUNX2 was firstly indicated on the sporadic patient of pedigree 2 through FISH, and then determined by CNV analysis.Conclusions:The sequenced heterozygous point mutation c.507delC in pedigree 1 caused a premature termination in RUNX2 protein. And in pedigree 2, the detected 3.5Mb large deletion caused a complete loss of RUNX2 function. These two novel genome variations are respectively etiological factors of two pedigrees. The identification of the novel mutations further expands the mutation spectrum, and will contribute to prenatal molecular diagnosis and preimplantation genetic diagnosis.更多还原