【作者】 李海燕；

【导师】 麻宏伟；

【作者基本信息】 中国医科大学 ， 儿科学， 2009， 博士

【摘要】 前言身材矮小是指身高处于同年龄、同性别正常儿童生长曲线第三百分位数以下或低于平均值2个标准差的儿童。根据外貌特征分为匀称性和非匀称矮小。非匀称性矮小又分为短肢矮小和短躯干矮小。身材是否匀称,是以坐高（顶臀长）与身高（长）的比例来判定的,坐高（顶臀长）占身高（长）的比例由出生时的0.67下降到14岁时的0.53,适应这一比例,为身材匀称。指间距反映上肢生长情况。正常时指距略小于身高。RaoE,报道矮小发病率为3%,国内资料报道发病率0.64%。身材矮小且伴有短肢畸形严重影响患儿的身体、生理及心理健康,遗传性骨病是这类疾病发病的重要原因也是目前研究的焦点。临床上导致短肢矮小的疾病有软骨发育不全（Achondroplasia,ACH）,干骺端软骨发育不良（MetaphysealChondrodysplasia Schmid type）,软骨生成不全（Achondrogenesis,ACG）、软骨外胚层发育不良（Ellis-Van Creveld syndrome,EVC）、成骨不全（Osteogenesisimperfecta,OI）、致死性发育不良（Thanatophoric dysplasia,TD）、窒息性胸廓发育不良（Asphxiating thoracic dyaplasia）、短肋-多指综合征（Short-rib polydactyliasyndrome）、假性软骨发育不全（Pseudoachondroplasia,PASACH）、软骨发育低下（Hypochondroplasia,HCH）及致密性骨发育障碍（Pycnodysostosis）等。这类疾病在临床上都以身材矮小并短肢畸形为共同表现,但各自又有其特征性表现。缺乏这类疾病临床特征的资料极大的限制了对这类疾病正确认识及诊断。从而进一步影响对这类疾病的深入研究。尤其是罕见疾病导致的短肢矮小如窒息性胸廓发育不良,致密性骨发育障碍等。目前国内仅有少数病种进行了基因水平的研究,如软骨发育不全的致病基因进行了研究报道。国外已经对多种导致短肢矮小的遗传性骨病进行了基因水平的研究,其中包括对致密性骨发育不良的致病基因组织溶酶体K（Cathepsin K,CTSK）的突变研究。国外还报道了在窒息性胸廓发育不良的患儿中发现了内纤毛转运蛋白80（intraflagellar transport80,IFT80）基因的突变。导致短肢矮小的病因国外多年前已经在基因水平上进行了发病机制的探讨。我国在这方面的研究还很少。由于许多疾病有着发病种族的差异性,对中国人自己的发病机制研究是必要的。尽管发病率低,但这类疾病严重影响患儿的生存质量,基因水平的研究有利于发病机制的研究,对优生优育具有重大意义。综上原因,我们对短肢矮小进行了临床特征分析。在临床特征分析的基础上,对中国致密性骨发育障碍一家系和窒息性胸廓发育不良一家系共两个家系进行了基因突变的研究。对象与方法1、对象2005年1月-2008年3月年在中国医科大学附属盛京医院发育儿科门诊因身材矮小并短肢畸形而就诊的患儿109例。年龄7个月～15岁,平均6.7岁。入选标准为身高处于同年龄、同性别正常儿童生长曲线第三百分位数以下或低于平均值2个标准差为矮小同时伴有短肢的患儿。对窒息性胸廓发育不良和致密性骨发育障碍家系分别进行IFT80基因和CTSK基因突变分析。2、方法1.首先对所有收集的短肢矮小病例进行临床特征分析。然后进行基因突变的研究。留取受试者外周静脉血3-5ml（ATD先证者留取脐血）,采用酚-氯仿-异戊醇法提取基因组DNA。根据Genbank中人类IFT80（intraflagellar transport protein,）基因序列,所有的编码外显子及外显子和内含子交界处设计引物,邻近的Exon17-18设计一对引物。引物设计采用Primer Premier 5.0软件,由Invitrogen生物公司合成,各引物序列、扩增片断长度及退火温度详见表1-1。根据Genbank中人类（CathepsinK）基因序列及参考文献。对所有的编码外显子及外显子和内含子交界处设计5对引物。其中外显子3-4及6-7各设计一对引物。常规PCR方法扩增目的片断,1.5%琼脂糖凝胶电泳检测PCR产物。对IFT80基因和CTSK基因的突变检测采用DNA直接测序的方法:PCR产物纯化后采用荧光素末端标记法(BigDye^TMTerminator),经ABI3730型全自动测序仪进行DNA测序。测序结果应用BLAST软件与GenBanK中人类IFT80和CTSK基因序列比对。结果1、通过对109例短肢矮小的临床特征的分析可以发现软骨发育不全的发病率最高,占30%（33/109）,窒息性胸廓发育不良和致密性骨发育障碍发病率最低各占0.9%（1/109）。2、骨龄异常占50.4%（55/109）;骨龄正常占49.6%（54/109）。3、致密性骨发育障碍的先证者CTSK基因为纯合性C/C突变核苷酸c.684（GenBank record:NM₀00396）.A＞C,导致蛋白p.Q187P的改变。4、致密性骨发育障碍的先证者父母CTSK基因在核苷酸c.684（GenBankrecord:NM₀00396）为杂合性A/C突变。5、在30例对照组中未发现CTSK基因存在该位点突变。6、在窒息性胸廓发育不良的先证者中未发现IFT80基因的编码区突变。结论1、通过对短肢矮小的临床特征分析可以提高对少见病的认识。提高正确诊断率,减少误诊的发生。2、通过对109例短肢矮小的临床特征的分析发现软骨发育不全的发病率最高,占30%（33/109）,窒息性胸廓发育不良和致密性骨发育障碍发病率最低各占0.9%（1/109）。3、骨龄异常占50.4%（55/109）;骨龄正常占49.6%（54/109）。骨龄评定对这类疾病的诊断价值有待进一步的研究。4、在中国致密性骨发育障碍的先证者中发现CTSK基因编码区突变,先证者为纯合性突变,其父母为杂合性突变。近亲结婚增加了发病风险,应该避免近亲结婚,降低发病率。5.在1例中国窒息性胸廓发育不良患儿中未发现IFT80基因的外显子编码区有突变。可能编码区的突变不是该病发病的唯一机制。中国患儿ATD的发病机制和IFT80基因的突变关系有待扩大样本量进一步明确。更多还原

【Abstract】 IntruductionMicrosomia is height under 3^rd percentile of growth curve or under 2 standard deviation of normal children.According to the characteristics of appearance,it was divided into symmetrical and unsymmetrical short stature.Unsymmetrical short stature were divided into short stature with short limb and short stature with short trunk. Whether the symmetry of stature is based on the proportion of sitting height（top of hip length） and height（Length） to determine.The morbidity of short stature had been reported about 3%by RaoE.And domestic data were about 0.64%.Short staure and skeletal deformity severely affected on physiological physical and psychological health. But there were few study on this directions.We described the condition of the disease, clinical features,X-rays features about Achondroplasia,Pseudoachondroplasia, osteogenesis imperfecta,Metaphyseal Chondrodysplasia（Schmid type）,Mesomelic dysplasia（Dyschondrosteosis）,Asphxiating thoracic dyaplasia and Pycnodysostosis,as a representative bone dysplasia which presents a short stature with short limbs. Analysis of clinic feature should provide the proof for diagnosis.However,Some diseases have similar clinical symptom.It’s difficulte to diagnose.Moulecular analysis would contribute to the differential diagnosis.Pycnodysostosis（MIM#265800） was first reported by Andren L in 1962.It is a rare autosomal recessive genetic bone abnormality caused by mutation of cathepsin K（CTSK）,which is the key enzyme for the degradation of the bone matrix protein by the osteoclasts.The disease affects a variety of ethnic groups.There were less than 200 patients described worldwide in the literature so far according to our knowledge.However,reports regarding CTSK mutations in Chinese with pycnodysostosis are scant.This is the first reported gene research about pycnodysostosis in Chinese.Jeune asphyxiating thoracic dystrophy （OMIM 208500）,an autosomal recessive chondrodysplasia,ofen leads to death in infancy because of a severely constricted thoracic cage and respiratory insufficiency. Beales PL had reported IFT80（intraflagellar transport） gene mutations in JATD.Up to date there are no simliar research in this directions.Despite the low morbidity,these diseases severly affect the live quality of relevant children.Systemic analysis of clinic feature should be beneficial to research pathogenesy.To sum up,we analysed clinical feature of short stature with short limb,in further study on mutational of CTSK and IFT80 gene in Chinese people with ATD and phycnodysosis.Materials and Methods1、SubjectsOne hundred nine children of Short stature with short limb of clinical features were analyzed.Among them age from 7 months to 15 years.They were recruited from the Clinic of Developmental Pediatrics of the Shengjing affiliated hospital of China Medical University between 2005 and 2008.To be selected by standard,which height were under 3rd percentile of the curve than the same year and homogeneity unimpaired child or under 2 standard deviation than general average and short limb.Molecular analysis of IFT80 and CTSK gene in Asphyxiating thoracic dysplasia and Pycnodysostosis respectively.2、Molecular analysis（1） Sample collection,preparation and extraction of DNABlood samples were collected after informed consent from the child’s parents. Genomic DNA was extracted form lymphocytes by using standard procedures.（2） Designation of the primersPrimers of CTSK gene were designed on the genomic sequence（GenBank-EMBL accession no.NC₀00001.8） by Primer Premier 5.0 software.A total of five amplimers including encoding exons and exon-intron boundaries were tested for DNA samples, exon3-4 and 6-7 being amplified simultaneously.Primers of IFT80 gene were designed on the genomic sequence（GenBank-EMBL accession no.NC₀00003.10 ）.A total of 19 amplimers including encoding exons and exon-intron boundaries were tested for DNA samples,exon17-18 being amplified simultaneously.All the primer were synthesized by Invitrongen Corporation（Shanghai branch office）.PCR product were detected by using 1.5%agarose gel electrophoresis.Then DNA sequencing was used by ABI3730 automatic sequencer.Sequencing results were compared with the reference human IFT80 and CTSK sequence.Results1.To analyze clinical features of 109 case short stature with short limb.ACH accountfor 30%（33/109） of all the 109 case.Asphyxiating thoracic dysplasia and Pycnodysostosis respective account for 0.9%（1/109）.Morbidity of ACH was the highest.Asphyxiating thoracic dysplasia and Pycnodysostosis was the lowest.2.Bone age abnormity account for 50.4%（55/109）;normity account for 49.6% （54/109）.3.Sequence analysis of all encoding exons and exon-intro boundaries sequences of the CTSK gene revealed that the proband had homozygous mutation of a single base A＞C transversion at nucleotide 684（GenBank record:NM₀00396）,which led to a Q187P amino acid change in the protein subsequently.4.The parents had heterozygous mutation.5.No similar changes in the CTSK gene sequence was observed in any of the controls.6.There were no mutation to be found in IFT80 gene in proband of ATD.Conclusion1.To analyze clinical features of Short stature with short limb should improved diagnoses rare disease and decrease misdiagnoses.2.To analyze clinical features of 109 case short stature with short limb.ACH account for 30%（33/109） of all the 109 case.Asphyxiating thoracic dysplasia and Pycnodysostosis respective account for 0.9%（1/109）.Morbidity of ACH was the highest.Asphyxiating thoracic dysplasia and Pycnodysostosis was the lowest.3.Bone age abnormity account for 50.4%（55/109）;normity account for 49.6% （54/109）.The value of bone age evaluation account for diagnose such disease need to further research.4.Mutations in the coding sequence of CTSK gene is a major pathogenesis in Chinese Pycnodysostosis patients.Proband was homozygosis mutation.His parents was heterozygosity mutation.Consanguineous marriage increase onset risk.So, Consanguineous marriage should be avoided. 5.There were no mutation to be found in the coding sequence of IFT80 gene in ATD.The coding region mutation should not be the only mechanisms of ATD.The relation between IFT80 gene and ATD should be further to study.更多还原