锁骨颅骨发育不良综合征患者的临床与基础研究
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摘要
锁骨颅骨发育不良综合征(Cleidocranial dysplasia,CCD)是一种少见的遗传性骨骼系统疾病，多为常染色体显性遗传方式。该综合征的致病基因为定位于染色体6p21的成骨细胞特异性转录因子（RUNX2/CBFa1），研究表明致病基因的碱基突变、大片段缺失、染色体异位、以及亚显微结构的变异是导致此综合征发生的分子学病因。锁骨颅骨发育不良可以影响膜性成骨、软骨成骨、以及牙齿的发育，其主要的临床表现为：锁骨的缺如/发育不全、囟门延迟闭合/开张、沃姆氏骨、恒牙迟萌、多生牙、以及其它骨骼异常。在所有临床表型中，因颅面部骨骼、牙齿的发育异常所造成的咀嚼功能障碍、面部畸形对患者的影响最大，是患者自觉发现的主要症状，也是患者就诊的主要原因。
目前，锁骨颅骨发育不良的临床工作重点、以及难点是对于该病的早期诊断以及错合畸形的矫治；研究工作的热点是应用分子生物学、生物信息学的技术、方法探讨分子学病因与机制。本研究通过对8例锁骨颅骨发育不良患者的颅颌面特征、以及治疗进行回顾性研究，为临床早期诊断的确立提供帮助，并指导临床开展序列治疗、以提高治疗的效果；对收集的4个锁骨颅骨发育不良家系进行RUNX2基因突变研究、以及蛋白结构、亚细胞定位功能的研究，以进一步探讨锁骨颅骨发育不良患者的发病原因，并为下一步开展遗传咨询、基因诊断以及产前诊断打下基础。本文内容分为三部分：
第一部分锁骨颅骨发育不良综合征患者的临床表型特征及序列治疗
对临床收集的来自4个不同家系的该病的疑似患者进行病史采集、详细的口腔检查、并结合X线检查了解患者全身骨骼、牙齿发育情况，以确立临床诊断。结果可见所有患者均具有颅骨、锁骨、牙齿发育异常的典型“三联征”表现，故临床明确诊断为锁骨颅骨发育不良综合征。所有患者的胸骨、骨盆、手、足也表现许多不典型的体征，充分说明此病是以颅骨、锁骨为主的广泛的骨骼发育异常。患者的临床表型变异性较大，在不同家系之间和同一家系的不同患者之间，都存在较大的表型差异。
为了全面认识锁骨颅骨发育不良患者的颅面部特征，对8例青少年患者的临床资料（面像、头颅定位侧位片、全口曲面断层片）进行回顾性分析。采用颈椎骨龄分析法，依据头颅定位侧位片的第二、三、四颈椎的形态分析患者的骨龄，结果显示：4例患者处于生长发育高峰前期CS-2或高峰期CS-3，其它4例患者的生长发育基本完成，处于CS-5或CS-6期。采用“三庭五眼”、“大三庭”、“小三庭”的面部比例划分标准，分析患者的面像，观察包括眼部（眼裂形态、眼眶距离），面部（突度、高度），额部（外形、突度）的颜面特征，结果显示：8例患者都表现眼裂倾斜，7例表现两眶距增宽；8例都表现前额部隆起，2例的前额正中出现凹陷；面中发育不足、面下高度比例不调在生长发育高峰期或前期的患者表现不明显，在生长发育基本完成的患者明显表现。通过全口曲面断层片、头颅定位侧位片观察包括下颌体部、下颌升支部、下颌角部的下颌特征，以及额骨、枕骨、鼻骨、颅底形态，并进行头影测量分析，结果显示：所有患者的喙突指向上方或后上方，下颌升支的前后缘向上平行/接近平行，枕骨出现沃姆氏骨，额骨不同程度隆起，下颌角不同程度缺失（程度严重者下颌形似“香蕉”），颅底蝶鞍部后凸；7例患者的鼻骨明显发育不足。头影测量分析显示：表示上下颌相对位置关系的测量指标（ANB角、Wits距、以及NA-PA）在生长发育完成的4例患者中都明显减小，在生长发育早期的4例患者中有轻度减小；所有患者的上颌长度、上颌位置、以及上面高、上面高与全面高的比值都减小，颌骨发育异常程度在生长发育完成的患者中表现较重。本研究说明除了上颌发育不足的面部异常未在生长发育早期的儿童明显表现外，其它部位的异常如多生牙，枕骨沃姆氏骨、鼻骨发育不足、喙突异常、眼距增宽等表现可在全部/大部患者中均有不同程度的表现，可为口腔医生对该病的早期诊断提供参考。
对患者治疗的回顾分析，可见患者伴有广泛的恒牙萌出异常、严重的错合畸形、以及颌骨发育异常；口腔治疗较为复杂、难度较大，治疗周期较长，需要口腔各学科的联合矫治。以恢复咀嚼功能、改善面部美观为目的，可以对锁骨颅骨发育不良的患者进行序列治疗，分为四个阶段：一期，大致年龄7-10岁，去除恒牙萌出障碍，定期观察牙齿萌出情况，待恒牙自发萌出；二期，大致年龄10-12岁，前牙牙根发育完成2/3，以促进前牙萌出、颌骨发育，为后续恒牙的萌出提供良好环境为目的，进行正畸牙齿矫治与早期颌骨矫形治疗。三期，大致年龄12-14岁，以促进尖牙/前磨牙萌出、颌骨发育，恢复牙齿功能为目的，进行正畸牙齿矫治与颌骨矫形治疗。四期，18-20岁，以纠正面部畸形，恢复口腔功能、美观为目的，进行成人正畸正颌联合治疗、以及义齿修复。虽然每一患者具体的矫治方案因人而异，但是对于此病的序列治疗目标、措施的制定，无疑对临床的治疗具有重要的指导意义。
第二部分锁骨颅骨发育不良综合征患者的基因诊断
对临床收集到的4个锁骨颅骨发育不良综合征家系进行聚合酶链式反应，DNA直接测序检测RUNX2基因突变，结果在1个家系的患者中未检测到RUNX2基因的位点突变；3个家系的患者中检测到不同的RUNX2基因的碱基突变，分别为：家系Ⅰ的突变体c.243-260del18（p.81-86del6）是在exon1的Q/A功能域的碱基缺失突变；家系Ⅱ的突变体c.1200C>A（p.stop400）是在exon7的NMTS区的终止密码子突变；家系Ⅲ的突变体c.674G>T（p.R225L）是位于exon3的NLS域的错义突变。其中，家系Ⅰ、Ⅱ的突变是本次研究中检测到的新的突变位点；家系Ⅲ的突变是国内外文献报道突变频率最高的位点。本研究结果进一步证明RUNX2基因的突变是锁骨颅骨发育不良的主要分子病因，并为国内外锁骨颅骨发育不良的突变位点数据库增添了新的资料。
对于RUNX2基因突变检测未发现突变位点的家系患者，通过实时定量PCR进行了拷贝数变异的检测，结果发现此家系患者RUNX2基因的全部外显子拷贝数明显降低，说明RUNX2基因的全部外显子缺失的杂合性突变是该家系发病的分子病因。本实验证明RUNX2基因亚显微结构的拷贝数变异是锁骨颅骨发育不良的又一分子病因；实时定量PCR方法操作简单、方便，结果敏感可靠，可以用于锁骨颅骨发育不良拷贝数变异的首选方法。
联合应用PCR-DNA测序和实时定量PCR两种分子遗传学检测方法，可以建立一个突变检测的技术平台，对于锁骨颅骨发育不良患者遗传学基因的诊断具有广阔的临床应用前景。第三部分基因突变对RUNX2蛋白三维构象与亚细胞定位影响的研究
为研究RUNX2基因的碱基突变对蛋白空间结构的影响，应用已有的生物信息学资源、借助相关生物信息学软件，对RUNX2基因的突变体蛋白结构进行了预测分析。Swiss-Model同源模建法预测，结果显示：错义突变体c.674G>T（p.R225L）的蛋白二级结构无明显改变，由于亲水性的精氨酸为疏水性的亮氨酸所替代，引起分子内氢键基团的丢失，以及分子表面静电势能的改变。I-TASSER综合模建法预测，结果显示：碱基缺失突变体c.243-260del18（p.81-86del6）由于发生6个氨基酸的缺失，使蛋白的二级和三级构象发生明显改变，丢失了部分螺旋及折迭结构；终止密码子突变体c.1200C>A（p.stop400），由于氨基酸的部分缺失、蛋白发生截短、出现明显的结构变化，丢失了所有的折迭结构以及大部分转角。本实验证明RUNX2突变体氨基酸序列的改变影响蛋白质的空间结构，从而可能改变蛋白的自身稳定性/功能、以及与其他蛋白质的相互作用，使蛋白的正常生理功能受到影响，最终导致锁骨颅骨发育不良的发生及发展。
为研究RUNX2基因突变体蛋白是否存在亚细胞定位功能的异常，通过构建包含突变体与野生型蛋白的真核表达载体，借助细胞转染，观察pEGFP-Nl-RUNX2载体转染组细胞的荧光蛋白表达情况，结果显示：在野生型RUNX2组的转染细胞中，绿色荧光蛋白仅在胞核中表达；引入突变位点的c.243-260del18（p.81-86del6）与c.1200C>A（p.stop400）两组，绿色荧光蛋白的表达情况与野生型相同，也是完全在胞核内表达；引入c.674G>T（p.R225L）的突变组转染细胞，在胞浆、胞核内均可见绿色荧光。本研究结果再次证明p.R225L对于RUNX2蛋白的核定位具有重要作用，表明p.81-86del6与p.stop400对于RUNX2蛋白的核定位没有影响，为进一步蛋白功能方面的研究提供实验依据。
Cleidocranial dysplasia (CCD) is a rare genetic disease of the skeletal system withautosomal dominant mode. The locus for CCD has been mapped to chromosome6p21where the osteoblast-specific transcription factor (RUNX2/CBFA1) has been located. Themolecular etiology of this syndrome is mutations of the base, large deletions,chromosome ectopic, and variation of submicroscopic structure. CCD can affect eitherthe ossification of membrane and cartilage, or the development of tooth. The mainclinical manifestations of CCD are absent or hypoplastic clavicles, persistently open ordelayed closure of sutures, wormian bones, delayed eruption of permanent dentition,supernumerary teeth, short stature, and other skeletal changes. Of all the clinicalphenotypes, craniofacial and dental abnormities which lead to chewing dysfunction andfacial deformity are the most conspicuous symptoms.
At present, early diagnosis of the disease and correction of tooth abnormities are themost important for clinicians; to explore the molecular etiology of the disease is the mostinteresting for researchers. In order to establish early diagnosis and to guide the clinical sequence therapy, retrospective study of eight cases of CCD was conducted. In order toinvestigate the pathogenesis of CCD and to carry out genetic counseling/geneticdiagnosis, the studies of RUNX2gene mutation and the function of the protein structurewere carried out for4unrelated pedigrees. This study consists of3parts, summarized asfollows:
1. Clinical phenotype and sequencing treatment of patients with cleidoeranialdysplasia
Detailed clinical examination and X-ray examination were undertaken for4unrelated pedigrees with cleidoeranial dysplasia. The results have shown that all patientshave typical "triad-performance", namely skull, clavicle, and teeth dysplasia, so thediagnosis of cleidoeranial dysplasia can be made. On the other hand, the abnormalities ofsternum, pelvis, hands, and feet also present in all of the patients with CCD. The skeletalabnormalities and oral manifestations of the syndrome are variable greatly betweendifferent families and different patients of the same family.
In order to fully understand craniofacial characteristics of the syndrome, the clinicaldata (facial photographs, lateral cephalometric radiographs, and panoramic radiographs)of eight cases with cleidoeranial dysplasia were retrospectively analyzed. To analysis theskeletal age of all the patients by lateral cephalometric radiographs, the results found that4patients were at the pre peak age (CS-2) or peak age (CS-3) of the growth anddevelopment;4patients finished the growth and development of skeleton (CS-5, orCS-6). The analysis of facial proportion, craniofacial characteristic has founded that8cases have inclined eye fissure, and7cases have wide orbital distances;8cases presentwith bulge forehead, and2cases have depression in the middle of the forehead; theunderdevelopment of middle faces of4pre-peak-age patients were more obvious thanthat of4post-development patients. Panoramic radiographs and lateral radiographs havefound that coracoids of all patients were pointed up or backwards; mandible angle,cranial base, and nasal bone of most cases are significantly abnormal. The maxillaryhypoplasia are not evident in the patients at the early growth and development, and other parts of abnormalities such as supernumerary teeth, occipital Worm's bone, nasal bonehypoplasia, and deformed coracoids may be presented in all/most of the patients.
The patients with CCD were affected with tooth eruption abnormalities, and severemalocclusion, so the oral treatment is very complicated and difficult. It is needed thecooperation of various disciplines to restore the chewing function and to improve thefacial appearance. The treatment of CCD can be divided into the following four stages:1)Approximately at7-10years old, it is needed to remove barriers to help the impactedteeth to erupt spontaneously;2) Approximately at13-14years old, it is needed to initiateorthodontic treatment to tract the impacted anterior tooth, and to provide a goodenvironment for the subsequent permanent teeth eruption;3) Approximately at13-14years old, for the purpose to restore tooth function, canines and premolars are neededorthodontic treatment;4) At18-20years old, orthognathic surgery is needed to correctfacial deformities for the purpose to restore the oral function and facial esthetic.2. Genetic diagnosis of patients with cleidocranial dysplasia
To identify mutations in the RUNX2gene, polymerase chain reaction and directsequencing of DNA were conducted for CCD cases came from4unrelated pedigrees.Beside one case of pedigree Ⅳ, three different mutations of RUNX2gene were detectedin CCD cases of the other pedigrees. Deletion mutation c.243-260del18(p.81-86del6)which was identified in pedigreeⅠis resided in Q/A domain of exon1; nonsensemutation c.1200C> A (p.stop400) which was identified in pedigreeⅡwas in the NMTSdomain of exon7; Mission mutant c.674G> T (p.R225L) which was identified inpedigree Ⅲ was located in the NLS domain of exon3. The mutations (p.81-86del6andp.stop400) of this study were detected for the first time, and the results of this studyfurther prove that RUNX2gene mutation is molecular etiology of CCD.
To identify copy number variation in RUNX2gene, real-time PCR was carried outin CCD case of pedigree Ⅳ. The results found that the copy number of all the exons ofRUNX2gene in this pedigree significantly decreased. This study has proved that copynumber variation is another molecular etiology of CCD; real-time PCR is a simple,convenient, sensitive and reliable method for the detection of copy number variation. Both PCR-DNA sequencing and real-time PCR can be conventionally used together tomake genetic diagnosis of patients with CCD.
3. The effect of mutations on three-dimensional structure and subcellularlocalization of RUNX2gene mutants
To analysis the protein structure of RUNX2gene mutants, bioinformatics softwarewere used to predict three-dimensional structure of the mutants. The results ofSwiss-Model homology modeling showed that the missense mutant c.674G> T (p.R225L)had no significant change in secondary structure; owning to the substitution of aminoacids, some intramolecular hydrogen bonding are lost, and the molecular electrostaticpotential energy is decreased. The results of I-TASSER modeling have showed that thesecondary and tertiary conformations of mutants (p.81-86del6and p.stop400) changesignificantly; parts of the helix and the folded structure miss. The experiment has provedthat the change of amino acid sequence affects the spatial structure of mutant proteins.
To ascertain the effect of mutations on subcellular localization of RUNX2protein,eukaryotic expression vectors containing the mutants and wild-type RUNX2wereconstructed. To observe the fluorescence of the pEGFP-Cl-RUNX2vector after celltransfection, the results found that green fluorescent protein expression of wild-type, andmutant(p.81-86del6, p.stop400) were completely expressed in the nucleus; mutantP.R225L was localized in both the cytoplasm and the nucleus. This experimentdemonstrated that nuclear localization of RUNX2protein was not affected by thep.81-86del6and p.stop400mutation.
目前，锁骨颅骨发育不良的临床工作重点、以及难点是对于该病的早期诊断以及错合畸形的矫治；研究工作的热点是应用分子生物学、生物信息学的技术、方法探讨分子学病因与机制。本研究通过对8例锁骨颅骨发育不良患者的颅颌面特征、以及治疗进行回顾性研究，为临床早期诊断的确立提供帮助，并指导临床开展序列治疗、以提高治疗的效果；对收集的4个锁骨颅骨发育不良家系进行RUNX2基因突变研究、以及蛋白结构、亚细胞定位功能的研究，以进一步探讨锁骨颅骨发育不良患者的发病原因，并为下一步开展遗传咨询、基因诊断以及产前诊断打下基础。本文内容分为三部分：
第一部分锁骨颅骨发育不良综合征患者的临床表型特征及序列治疗
对临床收集的来自4个不同家系的该病的疑似患者进行病史采集、详细的口腔检查、并结合X线检查了解患者全身骨骼、牙齿发育情况，以确立临床诊断。结果可见所有患者均具有颅骨、锁骨、牙齿发育异常的典型“三联征”表现，故临床明确诊断为锁骨颅骨发育不良综合征。所有患者的胸骨、骨盆、手、足也表现许多不典型的体征，充分说明此病是以颅骨、锁骨为主的广泛的骨骼发育异常。患者的临床表型变异性较大，在不同家系之间和同一家系的不同患者之间，都存在较大的表型差异。
为了全面认识锁骨颅骨发育不良患者的颅面部特征，对8例青少年患者的临床资料（面像、头颅定位侧位片、全口曲面断层片）进行回顾性分析。采用颈椎骨龄分析法，依据头颅定位侧位片的第二、三、四颈椎的形态分析患者的骨龄，结果显示：4例患者处于生长发育高峰前期CS-2或高峰期CS-3，其它4例患者的生长发育基本完成，处于CS-5或CS-6期。采用“三庭五眼”、“大三庭”、“小三庭”的面部比例划分标准，分析患者的面像，观察包括眼部（眼裂形态、眼眶距离），面部（突度、高度），额部（外形、突度）的颜面特征，结果显示：8例患者都表现眼裂倾斜，7例表现两眶距增宽；8例都表现前额部隆起，2例的前额正中出现凹陷；面中发育不足、面下高度比例不调在生长发育高峰期或前期的患者表现不明显，在生长发育基本完成的患者明显表现。通过全口曲面断层片、头颅定位侧位片观察包括下颌体部、下颌升支部、下颌角部的下颌特征，以及额骨、枕骨、鼻骨、颅底形态，并进行头影测量分析，结果显示：所有患者的喙突指向上方或后上方，下颌升支的前后缘向上平行/接近平行，枕骨出现沃姆氏骨，额骨不同程度隆起，下颌角不同程度缺失（程度严重者下颌形似“香蕉”），颅底蝶鞍部后凸；7例患者的鼻骨明显发育不足。头影测量分析显示：表示上下颌相对位置关系的测量指标（ANB角、Wits距、以及NA-PA）在生长发育完成的4例患者中都明显减小，在生长发育早期的4例患者中有轻度减小；所有患者的上颌长度、上颌位置、以及上面高、上面高与全面高的比值都减小，颌骨发育异常程度在生长发育完成的患者中表现较重。本研究说明除了上颌发育不足的面部异常未在生长发育早期的儿童明显表现外，其它部位的异常如多生牙，枕骨沃姆氏骨、鼻骨发育不足、喙突异常、眼距增宽等表现可在全部/大部患者中均有不同程度的表现，可为口腔医生对该病的早期诊断提供参考。
对患者治疗的回顾分析，可见患者伴有广泛的恒牙萌出异常、严重的错合畸形、以及颌骨发育异常；口腔治疗较为复杂、难度较大，治疗周期较长，需要口腔各学科的联合矫治。以恢复咀嚼功能、改善面部美观为目的，可以对锁骨颅骨发育不良的患者进行序列治疗，分为四个阶段：一期，大致年龄7-10岁，去除恒牙萌出障碍，定期观察牙齿萌出情况，待恒牙自发萌出；二期，大致年龄10-12岁，前牙牙根发育完成2/3，以促进前牙萌出、颌骨发育，为后续恒牙的萌出提供良好环境为目的，进行正畸牙齿矫治与早期颌骨矫形治疗。三期，大致年龄12-14岁，以促进尖牙/前磨牙萌出、颌骨发育，恢复牙齿功能为目的，进行正畸牙齿矫治与颌骨矫形治疗。四期，18-20岁，以纠正面部畸形，恢复口腔功能、美观为目的，进行成人正畸正颌联合治疗、以及义齿修复。虽然每一患者具体的矫治方案因人而异，但是对于此病的序列治疗目标、措施的制定，无疑对临床的治疗具有重要的指导意义。
第二部分锁骨颅骨发育不良综合征患者的基因诊断
对临床收集到的4个锁骨颅骨发育不良综合征家系进行聚合酶链式反应，DNA直接测序检测RUNX2基因突变，结果在1个家系的患者中未检测到RUNX2基因的位点突变；3个家系的患者中检测到不同的RUNX2基因的碱基突变，分别为：家系Ⅰ的突变体c.243-260del18（p.81-86del6）是在exon1的Q/A功能域的碱基缺失突变；家系Ⅱ的突变体c.1200C>A（p.stop400）是在exon7的NMTS区的终止密码子突变；家系Ⅲ的突变体c.674G>T（p.R225L）是位于exon3的NLS域的错义突变。其中，家系Ⅰ、Ⅱ的突变是本次研究中检测到的新的突变位点；家系Ⅲ的突变是国内外文献报道突变频率最高的位点。本研究结果进一步证明RUNX2基因的突变是锁骨颅骨发育不良的主要分子病因，并为国内外锁骨颅骨发育不良的突变位点数据库增添了新的资料。
对于RUNX2基因突变检测未发现突变位点的家系患者，通过实时定量PCR进行了拷贝数变异的检测，结果发现此家系患者RUNX2基因的全部外显子拷贝数明显降低，说明RUNX2基因的全部外显子缺失的杂合性突变是该家系发病的分子病因。本实验证明RUNX2基因亚显微结构的拷贝数变异是锁骨颅骨发育不良的又一分子病因；实时定量PCR方法操作简单、方便，结果敏感可靠，可以用于锁骨颅骨发育不良拷贝数变异的首选方法。
联合应用PCR-DNA测序和实时定量PCR两种分子遗传学检测方法，可以建立一个突变检测的技术平台，对于锁骨颅骨发育不良患者遗传学基因的诊断具有广阔的临床应用前景。第三部分基因突变对RUNX2蛋白三维构象与亚细胞定位影响的研究
为研究RUNX2基因的碱基突变对蛋白空间结构的影响，应用已有的生物信息学资源、借助相关生物信息学软件，对RUNX2基因的突变体蛋白结构进行了预测分析。Swiss-Model同源模建法预测，结果显示：错义突变体c.674G>T（p.R225L）的蛋白二级结构无明显改变，由于亲水性的精氨酸为疏水性的亮氨酸所替代，引起分子内氢键基团的丢失，以及分子表面静电势能的改变。I-TASSER综合模建法预测，结果显示：碱基缺失突变体c.243-260del18（p.81-86del6）由于发生6个氨基酸的缺失，使蛋白的二级和三级构象发生明显改变，丢失了部分螺旋及折迭结构；终止密码子突变体c.1200C>A（p.stop400），由于氨基酸的部分缺失、蛋白发生截短、出现明显的结构变化，丢失了所有的折迭结构以及大部分转角。本实验证明RUNX2突变体氨基酸序列的改变影响蛋白质的空间结构，从而可能改变蛋白的自身稳定性/功能、以及与其他蛋白质的相互作用，使蛋白的正常生理功能受到影响，最终导致锁骨颅骨发育不良的发生及发展。
为研究RUNX2基因突变体蛋白是否存在亚细胞定位功能的异常，通过构建包含突变体与野生型蛋白的真核表达载体，借助细胞转染，观察pEGFP-Nl-RUNX2载体转染组细胞的荧光蛋白表达情况，结果显示：在野生型RUNX2组的转染细胞中，绿色荧光蛋白仅在胞核中表达；引入突变位点的c.243-260del18（p.81-86del6）与c.1200C>A（p.stop400）两组，绿色荧光蛋白的表达情况与野生型相同，也是完全在胞核内表达；引入c.674G>T（p.R225L）的突变组转染细胞，在胞浆、胞核内均可见绿色荧光。本研究结果再次证明p.R225L对于RUNX2蛋白的核定位具有重要作用，表明p.81-86del6与p.stop400对于RUNX2蛋白的核定位没有影响，为进一步蛋白功能方面的研究提供实验依据。
Cleidocranial dysplasia (CCD) is a rare genetic disease of the skeletal system withautosomal dominant mode. The locus for CCD has been mapped to chromosome6p21where the osteoblast-specific transcription factor (RUNX2/CBFA1) has been located. Themolecular etiology of this syndrome is mutations of the base, large deletions,chromosome ectopic, and variation of submicroscopic structure. CCD can affect eitherthe ossification of membrane and cartilage, or the development of tooth. The mainclinical manifestations of CCD are absent or hypoplastic clavicles, persistently open ordelayed closure of sutures, wormian bones, delayed eruption of permanent dentition,supernumerary teeth, short stature, and other skeletal changes. Of all the clinicalphenotypes, craniofacial and dental abnormities which lead to chewing dysfunction andfacial deformity are the most conspicuous symptoms.
At present, early diagnosis of the disease and correction of tooth abnormities are themost important for clinicians; to explore the molecular etiology of the disease is the mostinteresting for researchers. In order to establish early diagnosis and to guide the clinical sequence therapy, retrospective study of eight cases of CCD was conducted. In order toinvestigate the pathogenesis of CCD and to carry out genetic counseling/geneticdiagnosis, the studies of RUNX2gene mutation and the function of the protein structurewere carried out for4unrelated pedigrees. This study consists of3parts, summarized asfollows:
1. Clinical phenotype and sequencing treatment of patients with cleidoeranialdysplasia
Detailed clinical examination and X-ray examination were undertaken for4unrelated pedigrees with cleidoeranial dysplasia. The results have shown that all patientshave typical "triad-performance", namely skull, clavicle, and teeth dysplasia, so thediagnosis of cleidoeranial dysplasia can be made. On the other hand, the abnormalities ofsternum, pelvis, hands, and feet also present in all of the patients with CCD. The skeletalabnormalities and oral manifestations of the syndrome are variable greatly betweendifferent families and different patients of the same family.
In order to fully understand craniofacial characteristics of the syndrome, the clinicaldata (facial photographs, lateral cephalometric radiographs, and panoramic radiographs)of eight cases with cleidoeranial dysplasia were retrospectively analyzed. To analysis theskeletal age of all the patients by lateral cephalometric radiographs, the results found that4patients were at the pre peak age (CS-2) or peak age (CS-3) of the growth anddevelopment;4patients finished the growth and development of skeleton (CS-5, orCS-6). The analysis of facial proportion, craniofacial characteristic has founded that8cases have inclined eye fissure, and7cases have wide orbital distances;8cases presentwith bulge forehead, and2cases have depression in the middle of the forehead; theunderdevelopment of middle faces of4pre-peak-age patients were more obvious thanthat of4post-development patients. Panoramic radiographs and lateral radiographs havefound that coracoids of all patients were pointed up or backwards; mandible angle,cranial base, and nasal bone of most cases are significantly abnormal. The maxillaryhypoplasia are not evident in the patients at the early growth and development, and other parts of abnormalities such as supernumerary teeth, occipital Worm's bone, nasal bonehypoplasia, and deformed coracoids may be presented in all/most of the patients.
The patients with CCD were affected with tooth eruption abnormalities, and severemalocclusion, so the oral treatment is very complicated and difficult. It is needed thecooperation of various disciplines to restore the chewing function and to improve thefacial appearance. The treatment of CCD can be divided into the following four stages:1)Approximately at7-10years old, it is needed to remove barriers to help the impactedteeth to erupt spontaneously;2) Approximately at13-14years old, it is needed to initiateorthodontic treatment to tract the impacted anterior tooth, and to provide a goodenvironment for the subsequent permanent teeth eruption;3) Approximately at13-14years old, for the purpose to restore tooth function, canines and premolars are neededorthodontic treatment;4) At18-20years old, orthognathic surgery is needed to correctfacial deformities for the purpose to restore the oral function and facial esthetic.2. Genetic diagnosis of patients with cleidocranial dysplasia
To identify mutations in the RUNX2gene, polymerase chain reaction and directsequencing of DNA were conducted for CCD cases came from4unrelated pedigrees.Beside one case of pedigree Ⅳ, three different mutations of RUNX2gene were detectedin CCD cases of the other pedigrees. Deletion mutation c.243-260del18(p.81-86del6)which was identified in pedigreeⅠis resided in Q/A domain of exon1; nonsensemutation c.1200C> A (p.stop400) which was identified in pedigreeⅡwas in the NMTSdomain of exon7; Mission mutant c.674G> T (p.R225L) which was identified inpedigree Ⅲ was located in the NLS domain of exon3. The mutations (p.81-86del6andp.stop400) of this study were detected for the first time, and the results of this studyfurther prove that RUNX2gene mutation is molecular etiology of CCD.
To identify copy number variation in RUNX2gene, real-time PCR was carried outin CCD case of pedigree Ⅳ. The results found that the copy number of all the exons ofRUNX2gene in this pedigree significantly decreased. This study has proved that copynumber variation is another molecular etiology of CCD; real-time PCR is a simple,convenient, sensitive and reliable method for the detection of copy number variation. Both PCR-DNA sequencing and real-time PCR can be conventionally used together tomake genetic diagnosis of patients with CCD.
3. The effect of mutations on three-dimensional structure and subcellularlocalization of RUNX2gene mutants
To analysis the protein structure of RUNX2gene mutants, bioinformatics softwarewere used to predict three-dimensional structure of the mutants. The results ofSwiss-Model homology modeling showed that the missense mutant c.674G> T (p.R225L)had no significant change in secondary structure; owning to the substitution of aminoacids, some intramolecular hydrogen bonding are lost, and the molecular electrostaticpotential energy is decreased. The results of I-TASSER modeling have showed that thesecondary and tertiary conformations of mutants (p.81-86del6and p.stop400) changesignificantly; parts of the helix and the folded structure miss. The experiment has provedthat the change of amino acid sequence affects the spatial structure of mutant proteins.
To ascertain the effect of mutations on subcellular localization of RUNX2protein,eukaryotic expression vectors containing the mutants and wild-type RUNX2wereconstructed. To observe the fluorescence of the pEGFP-Cl-RUNX2vector after celltransfection, the results found that green fluorescent protein expression of wild-type, andmutant(p.81-86del6, p.stop400) were completely expressed in the nucleus; mutantP.R225L was localized in both the cytoplasm and the nucleus. This experimentdemonstrated that nuclear localization of RUNX2protein was not affected by thep.81-86del6and p.stop400mutation.
引文
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