【作者】 刘立旻；

【导师】 夏维波；

【作者基本信息】 北京协和医学院 ， 临床医学， 2013， 博士

【摘要】 背景脊柱骨骺发育不良（spondyloepiphyseal dysplasia, SED）是一组罕见的以脊柱和骨骺畸形为特征的遗传性骨病,其遗传方式包括常染色体显性遗传、常染色体隐性遗传以及X染色体连锁隐性遗传。该病的共同特征是累及椎体和骨骺,临床上主要表现为短躯干型身材矮小、胸部畸形和关节退行性变。影像特点包括椎体扁平、骨骺发育不良及关节软骨破坏。依照《国际遗传性骨病分类标准（2010年版）》,可根据SED的临床特征、影像特点以及分子遗传学的不同表现分为九种临床类型：（1）先天性脊柱骨骺发育不良（spondyloepiphyseal dysplasia congenita, SEDC）;（2）迟发性脊柱骨骺发育不良（spondyloepiphyseal dysplasia tarda, SEDT）;（3）迟发性脊柱骨骺发育不良伴进行性骨关节病（spondyloepiphyseal dysplasia tarda with progressive arthropathy, SEDT-PA）;（4） Omani型SED（spondyloepiphyseal dysplasia Omani type, SED-OT）;（5） Kimberley型SED（spondyloepiphyseal dysplasia Kimberley type, SEDK）;（6） Wolcott-Rallison型SED（spondyloepiphyseal dysplasia Wolcott-Rallison type, SED-WR）;（7）轻度SED伴早发性关节炎（Mild SED with premature onset arthrosis）;（8）SED伴跖骨短缩畸形（SED with metatarsal shortening）;（9）常染色体隐性晚发型SED（Late onset SED, autosomal recessive type）..迄今,人类基因组突变数据库（HGMD Professional2012.3）收录的SED相关致病基因共8个,分别为COL2A1, TRAPPC2（SEDL）, WISP3, CHST3, ACAN（AGC1）, EIF2AK3, MATN3和PAPSS2,目前已有6个与SED临床类型相对应的致病基因得到了证实。虽然SED是罕见的遗传性骨病,其中较常见的临床类型的发病率尚不足2/100万,但SED所致的骨骼畸形不仅能影响患者的自身形象及正常运动能力,造成其生活质量下降及潜在的心理问题,该病还可能导致多系统受累,甚至威胁患者生命。因此,总结SED不同类型的临床表现并对其相关致病基因进行遗传学分析,有助于提高广大临床医师对该病的认识及诊断能力,也有望为选择国内热点突变进行中国人群SED致病基因建立者效应的相关研究,并藉此指导婚配,实施产前诊断,建立有效的基因诊断提供理论依据。目的1.对纳入本研究的尚未基因诊断SED的19个家系的临床资料进行总结分析。2.在所有临床诊断或拟诊SED的患者中筛查和检测8个能够导致SED的致病基因,确定SED患者的突变位点及突变类型。3.选取确诊SED的患者,对其相应的突变蛋白进行功能预测,并尝试探讨其临床表型与基因型的相关性。4.通过文献复习,对在中国人群中高发的SED类型进行临床表型及基因突变特点分析,以期为SED的临床分类以及分子遗传学诊断和评估提供证据支持,进一步丰富SED相关致病基因的突变数据库。方法1.研究对象：北京协和医院内分泌科自2006年至今收集的可以临床诊断或拟诊SED的19个家系。2.研究方法：（1）对19个SED家系先证者的遗传方式、临床表现及影像特点进行分析；（2）对19个SED家系先证者及其家系中有类似表现的亲属进行基因突变检测,针对8个可导致SED的致病基因（COL2A1, SEDL, WISP3, CHST3, AGC1, EIF2AK3, MATN3和PAPSS2）进行PCR扩增并测序,如发现新的错义突变则在50例无关健康对照中进行验证；（3）选取遗传学确诊的SED患者,应用SIFT和Polyphen-2进行突变体蛋白功能预测,并对其临床表型和基因型的相关性进行探讨；（4）通过文献复习,对在中国人群中高发的SED类型进行临床表型及基因突变特点分析。结果1.确诊SEDT-PA患者2例,其临床症状相似,均为男性,3-5岁起病,最初表现为双手指间关节肿大,逐渐累及双肘、髋、膝、踝等关节,存在关节疼痛和四肢多关节活动障碍。查体发现短躯干型身材矮小、脊柱侧弯、不同程度的步态异常及行走困难。影像学上均存在椎体扁平,椎体前缘不规则,四肢关节骨骺/干骺端增大及关节周围骨质疏松。基因检测证实了3个WISP3突变,其中c.624delA（K208fsX24）和c.105dupT （G36fsX10）为新发现的突变,c.342T>G （C114W）为国内多次报道的已知突变。文献复习发现SEDT-PA是随年龄增加呈进行性进展的疾病,患者起病越早,病程越长,临床表现可能越严重。c.866dupA, c.1000T>C, c.342T>G和c.341G>A存在于多个无血缘关系的中国SEDT-PA家系,提示在中国人群中可能存在WISP3基因的建立者效应。2.确诊SEDT患者2例,其临床症状相似,均为男性,儿童期起病,以短躯干型身材矮小为特征。影像学方面表现为椎体扁平,椎体前部上下缘凹陷、中后部呈特征性的“驼峰状”突起。基因检测证实了2个SEDL突变,分别为无义突变c.364C>T（R122X）和缺失突变c.191₁92delTG （G64fsX24）,两者均为先前报道过的重复突变。3.确诊SEDC患者1例,为女性,自幼起病,临床上表现为明显的短躯干型身材矮小、鸭步步态、膝外翻、足外翻和脊柱侧弯。影像学上存在胸腰椎椎体扁平,密度减低,多发椎体楔形变。基因检测证实了COL2A1基因错义突变c.3446₃447GA>TC（G1149V）,此为新发现的突变。应用SIFT和Polyphen-2对突变体蛋白进行功能预测显示该突变可严重损害II型胶原的正常功能。结论对纳入本研究的19例可以临床诊断或拟诊SED的患者及其家系进行临床资料分析总结,通过PCR技术在每位患者中逐一筛查和检测了8个能够导致SED的致病基因,最终确诊了SEDT-PA患者两例,SEDT患者两例,以及SEDC患者一例。共发现致病基因突变6个,其中WISP3基因突变3个,分别为c.624delA（K208fsX24）, c.105dupT（G36fsX10）和c.342T>G （C114W）; SEDL基因突变2个,分别为C.364C>T （R122X）和c.191₁92delTG （G64fsX24）;以及1个COL2A1基因突变c.3446₃447GA>TC（G1149V）。c.624delA （K208fsX24）,c.105dupT （G36fsX10）和c.3446₃447GA>TC（G1149V）为目前尚未报道的新突变。对错义突变c.342T>G（C114W）和c.3446₃447GA>TC（G1149V）进行蛋白功能预测分析,结果显示这些突变均可严重损害相关编码蛋白的正常功能。本研究进一步在中国人群中证实了COL2A1、SEDL和WISP3基因是可以导致SED的致病基因,丰富了SED相关致病基因的突变数据库。更多还原

【Abstract】 IntroductionSpondyloepiphyseal dysplasia （SED） is a group of rare genetic skeletal disorder, which predominantly involves the spine and epiphysis, with the leading features of disproportionately short statue, thoracocyllosis, and progressive degeneration of multiple joints. Radiographic findings usually display platyspondyly, epiphyseal dysplasia and narrowed joint spaces. The inheritance pattern of SED consists of autosomal dominant, autosomal recessive and X-linked recessive modes.According to the nosology and classification of genetic skeletal disorders （2010revision）, SED can be further divided into nine distinct clinical categories based on clinical manifestations, radiographic features, as well as characteristics of molecular genetics. The nine clinical types of SED include:（1） spondyloepiphyseal dysplasia congenita （SEDC）;（2） spondyloepiphyseal dysplasia tarda （SEDT）;（3） spondyloepiphyseal dysplasia tarda with progressive arthropathy （SEDT-PA）;（4） spondyloepiphyseal dysplasia Omani type （SED-OT）;（5） spondyloepiphyseal dysplasia Kimberley type （SEDK）;（6） spondyloepiphyseal dysplasia Wolcott-Rallison type （SED-WR）;（7） Mild SED with premature onset arthrosis;（8） SED with metatarsal shortening;（9） late onset SED, autosomal recessive type.To date, a total of eight different genes that are associated with SED （COL2A1, SEDL, WISP3, CHST3, AGC1, EIF2AK3, MATN3and PAPSS2） have been included in the Human Gene Mutation Database （HGMD Professional2012.3）, six of which have been identified to be responsible for the pathogenesis of the corresponding clinical types of SED.Objective1. To analyze the clinical manifestations and expression profiles of the19recruited families of suspected or clinically diagnosed SED.2. To perform the genetic analysis in all the patients by detecting eight pathogenic genes of SED, and confirm the precise mutant site and type of each mutation. 3. To predict the protein functions of all the missense mutations detected in this study, and try to explore their correlation between phenotype and genotype.4. To summarize the current knowledge about the spectrum of SED phenotype and corresponding genetic mutations in the Chinese population.Methods1. Subjects:19recruited families of suspected or clinically diagnosed SED in the Department of Endocrinology of PUMCH since2006.2. Methods:（1） We analyzed the inheritance patterns, clinical manifestations and radiographic features of the probands from19recruited families of suspected or clinically diagnosed SED.（2） Using PCR technique, we amplified the COL2A1, SEDL, WISP3, CHST3, AGC1, EIF2AK3, MATN3and PAPSS2gene fragments of the19probands,conducted the direct nucleotide sequencing of all the PCR products, and confirmed the novel missense mutations in50ethnically matched controls.（3） The protein functions of the all the missense mutations were predicted by SIFT and Polyphen-2.（4） We analyzed and summarized the current knowledge about the spectrum of SED phenotype and corresponding genetic mutations in the Chinese population through literature review.Results1. Two SEDT-PA patients were genetically diagnosed in our study. Both of them were male and shared the similar clinical features. The joint swelling of the patients started at the age of3to5, first appearing in the bilateral interphalangeal joints, then subsequently in elbows, hips, knees and ankles, with remarkable articular pain and mobility limitation. Upon physical examination, the patients were noted to have disproportionately short stature, scoliosis, gait disturbance and difficulties in ambulation. Radiographic findings revealed platyspondyly, widened epiphyses, narrowed joint spaces and general osteoporosis. Three WISP3mutations were identified by genetic analysis, among which, c.624delA （K208fsX24） and c.105dupT （G36fsX10） were novel mutations, c.342T>G （C114W） has been previously reported as of Chinese origin. Literature review showed SEDT-PA was a progressive disorder, and that patients with relatively earlier onsets might result in severer clinical phenotypes. The WISP3gene mutations c.866dupA, c.1000T>C, c.342T>G and c.341G>A have repeatedly presented in distinct Chinese pedigrees, and family investigations have revealed a considerable number of mutation carriers, probably suggesting the existence of a founder effect among the Chinese population.2. Two male SEDT patients were genetically diagnosed in our study. The initial symptom of the disorder emerged in their childhood years. Disproportionately short stature was the leading clinical feature of both patients. Radiographic findings revealed platyspondyly and the typical "posterior hump" of the vertebral bodies. Two recurrent SEDL mutations were detected by genetic analysis:nonsense mutation c.364C>T （R122X） and deletion mutation c.191₁92delTG （G64fsX24）.3. One female SEDC patient was genetically diagnosed in our study as well. She was characterized by congenital dwarfism with a short trunk, scoliosis, genu valgus, talipes valgus and gait disturbance. Radiographic findings showed platyspondyly, scoliosis and anterior wedging of the vertebral bodies. Genetic analysis revealed a novel missense mutation c.3446₃447GA>TC （G1149V） in the49th exon of the COL2A1gene. Protein prediction of this missense mutation by SIFT and Polyphen-2has indicated severe damage to the function of type Ⅱ collagen.ConclusionIn the current study, we analyzed the clinical data of the probands from19recruited families of suspected or clinically diagnosed SED. Genetic studies of the pathogenic genes relevant to SED revealed a total number of6mutations, including three novel mutations:c.624delA （K208fsX24）, c.105dupT （G36fsX10） and c.3446₃447GA>TC （G1149V）. Our findings contributed to the further expansion of the original mutation spectrum of SED, and demonstrated that the mutations of COL2A1, SEDL, and WISP3were pathogenic for the development of SED in the Chinese population.更多还原