本文關(guān)鍵詞：鎖骨顱骨發(fā)育不良綜合征患者的臨床與基礎(chǔ)研究，由筆耕文化傳播整理發(fā)布。

        鎖骨顱骨發(fā)育不良綜合征(Cleidocranial dysplasia,CCD)是一種少見的遺傳性骨骼系統(tǒng)疾病，多為常染色體顯性遺傳方式。該綜合征的致病基因為定位于染色體6p21的成骨細(xì)胞特異性轉(zhuǎn)錄因子（RUNX2/CBFa1），研究表明致病基因的堿基突變、大片段缺失、染色體異位、以及亞顯微結(jié)構(gòu)的變異是導(dǎo)致此綜合征發(fā)生的分子學(xué)病因。鎖骨顱骨發(fā)育不良可以影響膜性成骨、軟骨成骨、以及牙齒的發(fā)育，其主要的臨床表現(xiàn)為：鎖骨的缺如/發(fā)育不全、囟門延遲閉合/開張、沃姆氏骨、恒牙遲萌、多生牙、以及其它骨骼異常。在所有臨床表型中，因顱面部骨骼、牙齒的發(fā)育異常所造成的咀嚼功能障礙、面部畸形對患者的影響最大，是患者自覺發(fā)現(xiàn)的主要癥狀，也是患者就診的主要原因。目前，鎖骨顱骨發(fā)育不良的臨床工作重點、以及難點是對于該病的早期診斷以及錯合畸形的矯治；研究工作的熱點是應(yīng)用分子生物學(xué)、生物信息學(xué)的技術(shù)、方法探討分子學(xué)病因與機制。本研究通過對8例鎖骨顱骨發(fā)育不良患者的顱頜面特征、以及治療進(jìn)行回顧性研究，為臨床早期診斷的確立提供幫助，并指導(dǎo)臨床開展序列治療、以提高治療的效果；對收集的4個鎖骨顱骨發(fā)育不良家系進(jìn)行RUNX2基因突變研究、以及蛋白結(jié)構(gòu)、亞細(xì)胞定位功能的研究，以進(jìn)一步探討鎖骨顱骨發(fā)育不良患者的發(fā)病原因，并為下一步開展遺傳咨詢、基因診斷以及產(chǎn)前診斷打下基礎(chǔ)。本文內(nèi)容分為三部分：第一部分鎖骨顱骨發(fā)育不良綜合征患者的臨床表型特征及序列治療對臨床收集的來自4個不同家系的該病的疑似患者進(jìn)行病史采集、詳細(xì)的口腔檢查、并結(jié)合X線檢查了解患者全身骨骼、牙齒發(fā)育情況，以確立臨床診斷。結(jié)果可見所有患者均具有顱骨、鎖骨、牙齒發(fā)育異常的典型“三聯(lián)征”表現(xiàn)，故臨床明確診斷為鎖骨顱骨發(fā)育不良綜合征。所有患者的胸骨、骨盆、手、足也表現(xiàn)許多不典型的體征，充分說明此病是以顱骨、鎖骨為主的廣泛的骨骼發(fā)育異常。患者的臨床表型變異性較大，在不同家系之間和同一家系的不同患者之間，都存在較大的表型差異。為了全面認(rèn)識鎖骨顱骨發(fā)育不良患者的顱面部特征，對8例青少年患者的臨床資料（面像、頭顱定位側(cè)位片、全口曲面斷層片）進(jìn)行回顧性分析。采用頸椎骨齡分析法，依據(jù)頭顱定位側(cè)位片的第二、三、四頸椎的形態(tài)分析患者的骨齡，結(jié)果顯示：4例患者處于生長發(fā)育高峰前期CS-2或高峰期CS-3，其它4例患者的生長發(fā)育基本完成，處于CS-5或CS-6期。采用“三庭五眼”、“大三庭”、“小三庭”的面部比例劃分標(biāo)準(zhǔn)，分析患者的面像，觀察包括眼部（眼裂形態(tài)、眼眶距離），面部（突度、高度），額部（外形、突度）的顏面特征，結(jié)果顯示：8例患者都表現(xiàn)眼裂傾斜，7例表現(xiàn)兩眶距增寬；8例都表現(xiàn)前額部隆起，2例的前額正中出現(xiàn)凹陷；面中發(fā)育不足、面下高度比例不調(diào)在生長發(fā)育高峰期或前期的患者表現(xiàn)不明顯，在生長發(fā)育基本完成的患者明顯表現(xiàn)。通過全口曲面斷層片、頭顱定位側(cè)位片觀察包括下頜體部、下頜升支部、下頜角部的下頜特征，以及額骨、枕骨、鼻骨、顱底形態(tài)，并進(jìn)行頭影測量分析，結(jié)果顯示：所有患者的喙突指向上方或后上方，下頜升支的前后緣向上平行/接近平行，枕骨出現(xiàn)沃姆氏骨，額骨不同程度隆起，下頜角不同程度缺失（程度嚴(yán)重者下頜形似“香蕉”），顱底蝶鞍部后凸；7例患者的鼻骨明顯發(fā)育不足。頭影測量分析顯示：表示上下頜相對位置關(guān)系的測量指標(biāo)（ANB角、Wits距、以及NA-PA）在生長發(fā)育完成的4例患者中都明顯減小，在生長發(fā)育早期的4例患者中有輕度減��；所有患者的上頜長度、上頜位置、以及上面高、上面高與全面高的比值都減小，頜骨發(fā)育異常程度在生長發(fā)育完成的患者中表現(xiàn)較重。本研究說明除了上頜發(fā)育不足的面部異常未在生長發(fā)育早期的兒童明顯表現(xiàn)外，其它部位的異常如多生牙，枕骨沃姆氏骨、鼻骨發(fā)育不足、喙突異常、眼距增寬等表現(xiàn)可在全部/大部患者中均有不同程度的表現(xiàn)，可為口腔醫(yī)生對該病的早期診斷提供參考。對患者治療的回顧分析，可見患者伴有廣泛的恒牙萌出異常、嚴(yán)重的錯合畸形、以及頜骨發(fā)育異常；口腔治療較為復(fù)雜、難度較大，治療周期較長，需要口腔各學(xué)科的聯(lián)合矯治。以恢復(fù)咀嚼功能、改善面部美觀為目的，可以對鎖骨顱骨發(fā)育不良的患者進(jìn)行序列治療，分為四個階段：一期，大致年齡7-10歲，去除恒牙萌出障礙，定期觀察牙齒萌出情況，待恒牙自發(fā)萌出；二期，大致年齡10-12歲，前牙牙根發(fā)育完成2/3，以促進(jìn)前牙萌出、頜骨發(fā)育，為后續(xù)恒牙的萌出提供良好環(huán)境為目的，進(jìn)行正畸牙齒矯治與早期頜骨矯形治療。三期，大致年齡12-14歲，以促進(jìn)尖牙/前磨牙萌出、頜骨發(fā)育，恢復(fù)牙齒功能為目的，進(jìn)行正畸牙齒矯治與頜骨矯形治療。四期，18-20歲，以糾正面部畸形，恢復(fù)口腔功能、美觀為目的，進(jìn)行成人正畸正頜聯(lián)合治療、以及義齒修復(fù)。雖然每一患者具體的矯治方案因人而異，但是對于此病的序列治療目標(biāo)、措施的制定，無疑對臨床的治療具有重要的指導(dǎo)意義。第二部分鎖骨顱骨發(fā)育不良綜合征患者的基因診斷對臨床收集到的4個鎖骨顱骨發(fā)育不良綜合征家系進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)，DNA直接測序檢測RUNX2基因突變，結(jié)果在1個家系的患者中未檢測到RUNX2基因的位點突變；3個家系的患者中檢測到不同的RUNX2基因的堿基突變，分別為：家系Ⅰ的突變體c.243-260del18（p.81-86del6）是在exon1的Q/A功能域的堿基缺失突變；家系Ⅱ的突變體c.1200C>A（p.stop400）是在exon7的NMTS區(qū)的終止密碼子突變；家系Ⅲ的突變體c.674G>T（p.R225L）是位于exon3的NLS域的錯義突變。其中，家系Ⅰ、Ⅱ的突變是本次研究中檢測到的新的突變位點；家系Ⅲ的突變是國內(nèi)外文獻(xiàn)報道突變頻率最高的位點。本研究結(jié)果進(jìn)一步證明RUNX2基因的突變是鎖骨顱骨發(fā)育不良的主要分子病因，并為國內(nèi)外鎖骨顱骨發(fā)育不良的突變位點數(shù)據(jù)庫增添了新的資料。對于RUNX2基因突變檢測未發(fā)現(xiàn)突變位點的家系患者，通過實時定量PCR進(jìn)行了拷貝數(shù)變異的檢測，結(jié)果發(fā)現(xiàn)此家系患者RUNX2基因的全部外顯子拷貝數(shù)明顯降低，說明RUNX2基因的全部外顯子缺失的雜合性突變是該家系發(fā)病的分子病因。本實驗證明RUNX2基因亞顯微結(jié)構(gòu)的拷貝數(shù)變異是鎖骨顱骨發(fā)育不良的又一分子病因；實時定量PCR方法操作簡單、方便，結(jié)果敏感可靠，可以用于鎖骨顱骨發(fā)育不良拷貝數(shù)變異的首選方法。聯(lián)合應(yīng)用PCR-DNA測序和實時定量PCR兩種分子遺傳學(xué)檢測方法，，可以建立一個突變檢測的技術(shù)平臺，對于鎖骨顱骨發(fā)育不良患者遺傳學(xué)基因的診斷具有廣闊的臨床應(yīng)用前景。第三部分基因突變對RUNX2蛋白三維構(gòu)象與亞細(xì)胞定位影響的研究為研究RUNX2基因的堿基突變對蛋白空間結(jié)構(gòu)的影響，應(yīng)用已有的生物信息學(xué)資源、借助相關(guān)生物信息學(xué)軟件，對RUNX2基因的突變體蛋白結(jié)構(gòu)進(jìn)行了預(yù)測分析。Swiss-Model同源模建法預(yù)測，結(jié)果顯示：錯義突變體c.674G>T（p.R225L）的蛋白二級結(jié)構(gòu)無明顯改變，由于親水性的精氨酸為疏水性的亮氨酸所替代，引起分子內(nèi)氫鍵基團的丟失，以及分子表面靜電勢能的改變。I-TASSER綜合模建法預(yù)測，結(jié)果顯示：堿基缺失突變體c.243-260del18（p.81-86del6）由于發(fā)生6個氨基酸的缺失，使蛋白的二級和三級構(gòu)象發(fā)生明顯改變，丟失了部分螺旋及折迭結(jié)構(gòu)；終止密碼子突變體c.1200C>A（p.stop400），由于氨基酸的部分缺失、蛋白發(fā)生截短、出現(xiàn)明顯的結(jié)構(gòu)變化，丟失了所有的折迭結(jié)構(gòu)以及大部分轉(zhuǎn)角。本實驗證明RUNX2突變體氨基酸序列的改變影響蛋白質(zhì)的空間結(jié)構(gòu)，從而可能改變蛋白的自身穩(wěn)定性/功能、以及與其他蛋白質(zhì)的相互作用，使蛋白的正常生理功能受到影響，最終導(dǎo)致鎖骨顱骨發(fā)育不良的發(fā)生及發(fā)展。為研究RUNX2基因突變體蛋白是否存在亞細(xì)胞定位功能的異常，通過構(gòu)建包含突變體與野生型蛋白的真核表達(dá)載體，借助細(xì)胞轉(zhuǎn)染，觀察pEGFP-Nl-RUNX2載體轉(zhuǎn)染組細(xì)胞的熒光蛋白表達(dá)情況，結(jié)果顯示：在野生型RUNX2組的轉(zhuǎn)染細(xì)胞中，綠色熒光蛋白僅在胞核中表達(dá)；引入突變位點的c.243-260del18（p.81-86del6）與c.1200C>A（p.stop400）兩組，綠色熒光蛋白的表達(dá)情況與野生型相同，也是完全在胞核內(nèi)表達(dá)；引入c.674G>T（p.R225L）的突變組轉(zhuǎn)染細(xì)胞，在胞漿、胞核內(nèi)均可見綠色熒光。本研究結(jié)果再次證明p.R225L對于RUNX2蛋白的核定位具有重要作用，表明p.81-86del6與p.stop400對于RUNX2蛋白的核定位沒有影響，為進(jìn)一步蛋白功能方面的研究提供實驗依據(jù)。

    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 cleidoeranialdysplasiaDetailed 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 dysplasiaTo 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 mutantsTo 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.

鎖骨顱骨發(fā)育不良綜合征患者的臨床與基礎(chǔ)研究

縮略語表5-7中文摘要7-11Abstract11-14前言15-18文獻(xiàn)回顧18-33第一部分 鎖骨顱骨發(fā)育不良綜合征患者的臨床表型特征及序列治療33-49    實驗一 鎖骨顱骨發(fā)育不良綜合征患者的臨床檢查與分析33-40        1 材料33-34        2 方法34        3 結(jié)果34-37        4 討論37-40    實驗二 鎖骨顱骨發(fā)育不良綜合征患者的顱面部特征分析及治療策略40-49        1 材料40        2 方法40-41        3 結(jié)果41-45        4 討論45-49第二部分 鎖骨顱骨發(fā)育不良綜合征患者的基因診斷49-65    實驗一 鎖骨顱骨發(fā)育不良綜合征患者的致病基因突變位點的檢測49-58        1 材料49-50        2 方法50-52        3 結(jié)果52-55        4 討論55-58    實驗二 鎖骨顱骨發(fā)育不良綜合征患者的致病基因拷貝數(shù)變異的檢測58-65        1 材料58        2 方法58-60        3 結(jié)果60-62        4 討論62-65第三部分 基因突變對 RUNX2 蛋白三維構(gòu)象與亞細(xì)胞定位影響的研究65-79    實驗一 RUNX2 基因突變體蛋白三維構(gòu)象的計算機預(yù)測分析65-71        1 材料65        2 方法65-66        3 結(jié)果66-68        4 討論68-71    實驗二 基因突變對 RUNX2 蛋白亞細(xì)胞定位的影響71-79        1 材料71        2 方法71-75        3 結(jié)果75        4 討論75-79小結(jié)79-81參考文獻(xiàn)81-91附錄91-99臨床病例報告99-115個人簡歷和研究成果115-116致謝116

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