【作者】 赵军；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学， 2007， 博士

【摘要】 目的：收集先天性眼外肌纤维化(congenital fibrosis of the extraocular muscles,CFEOM)家系及散发病例，分析其临床表型特征和分子遗传学病因，对CFEOM家系进行基因连锁分析定位和候选基因KIF21A的突变检测，对散发病例直接进行基因KIF21A序列的突变筛查。研究先天性眼外肌纤维化的分子发病机制、病理特点及其与临床表型间的关系。1研究CFEOM家系及散发病例的临床表现、眼外肌病理和超微结构及眼眶CT特征。2基因定位：选取分布在12号染色体FEOM1区域的多态性位点并对CFEOM家系患者进行连锁分析。3基因序列分析：采取直接DNA序列分析对CFEOM家系成员及散发患者进行KIF21A基因的突变检测。4单链构象多态性分析(single-stranded conformational polymorphism,SSCP)：证实突变，并排除是多态性的可能。方法：1临床研究1．1 CFEOM家系患者及散发病例病史采集，分别进行视力、屈光状态、裂隙灯、眼底镜、眼位、眼球运动和提上睑肌肌力等检查。1．2对CFEOM家系患者及散发患者在手术中所取眼外肌组织进行病理及超微结构研究。正常对照选自因眼外伤行眼球摘除术的无眼球运动障碍病史的患者的眼外肌组织。1．3对CFEOM患者及正常人作为对照进行眼眶CT检测。2分子遗传学研究2．1取CFEOM家系成员及散发患者外周静脉血5-8ml，采用Roche Biochimical公司DNA分离试剂盒提取基因组DNA。2．2基因连锁分析选取分布在第12号染色体FEOM1区域的6对微卫星多态标记并设计合成引物，利用聚合酶链反应(Polymerase Chain Reaction PCR)扩增微卫星标记物，并利用ABI3130-avant全自动遗传分析仪读取6对微卫星标记物的等位基因片段大小，应用Genescan 3.7软件和Genetyper 3.7软件进行单体型结构分析，利用LINKAGE 5.1软件进行两点法计算LOD值并构建单体型。2．3基因序列分析采取直接DNA序列分析，将CFEOM家系患者及散发病例的KIF21A基因的外显子8，20及21的PCR扩增产物在ABI 3130-avant全自动遗传分析仪电泳并进行基因序列分析。如发现可能的基因突变则通过SSCP证实突变，并在相关人群中进行筛查。结果：1临床研究1．1临床表现该CFEOM家系四代共49人，14名患者，具有完全外显的常染色体显性遗传特征，为CFEOM1型，患者临床表现的共同特征为双眼位于下斜位，上转不到中线，双眼上睑下垂。不同之处为垂直眼位、水平眼位及眼球运动限制各异，部分患者伴有瞳孔缩小和异常眼球运动，该家系最突出的一个特征为14名患者中有11名患者合并少年白发。该合并症在检索的的所有文献中尚未见报道。13名散发患者中有一名患者表现为单眼CFEOM，另一名患者双眼存在明显差异，一眼眼位居中，上转受限，另一眼位于下斜位，上转不能过中线，并存在上睑下垂，这两名患者的表型符合CFEOM3型。其余11名散发患者均表现为双眼位于下斜位，上转不到中线，双眼上睑下垂。部分患者伴有异常眼球运动、下睑内翻、牙齿发育不良及视神经发育不良等合并症。手术中行被动牵拉试验所有患者均存在明显限制，眼外肌存在不同程度的僵硬、失去弹性及萎缩。治疗主要包括矫正屈光不正治疗弱视及手术治疗斜视和上睑下垂，斜视矫正主要为下直肌后徙矫正下斜视，水平肌肉的后徙／缩短术矫正水平斜视。上睑下垂可根据提上睑肌的力量情况通过提上睑肌缩短术或额肌悬吊术加以矫正。1．2患者眼外肌病理显示肌纤维粗细不均，部分肌纤维萎缩变细，肌细胞核增多，在肌纤维间充填有较多的原纤维样物质。超微结构研究显示眼外肌肌节不清晰，线粒体聚集，部分空泡化。1．3眼眶CT检查示所有CFEOM患者上直肌和提上睑肌较正常对照明显变细或缺如，部分CFEOM患者的内直肌、外直肌或上斜肌有不同程度变细，下直肌未见明显异常。所有CFEOM患者视神经与正常对照相比向鼻上移位，且部分患者视神经较细小。2分子遗传学研究对12号染色体显性遗传位点进行连锁分析，发现在12号染色体FEOM1位点附近的多态性标记D12S1048、D12S1663、D12S85、D12S1661、D12S1635及D12S398与患者共分离，利用两点法计算微卫星标记物连锁分析LOD值，其中在D12S1048取得最大LOD值为3.41。提示致病基因可能为KIF21A。对KIF21A基因直接测序显示家系中所有患者在KIF21A基因的第21外显子存在杂合性突变，即KIF21A在2860位点发生了C→T的碱基突变，该替换导致第954位密码子由CGG→TGG，使精氨酸变为色氨酸(R954W)，并且该突变与疾病表型共分离。954位氨基酸精氨酸在进化中是高度保守的。SSCP分析证实了该突变，而家系未受累者和100名正常对照未检测到这一突变。说明KIF21A基因突变为该CFEOM家系患者的致病基因。散发患者中有6名患者存在KIF21A基因的第21外显子的杂合性突变，即KIF21A在2860位点发生了C→T的碱基突变。结论：(1)我们确定了一个常染色体显性遗传的CFEOM1家系，家系成员共49人，14名患者。同时确定13名CFEOM散发患者。(2)经连锁分析将致病基因定位于12号染色体FEOM1位点，在微卫星多态性标记D12S1048取得最大LOD值为3.41。(3)测序发现CFEOM1家系患者存在KIF21A基因外显子21的c．2860C→T突变，SSCP分析证实了这一突变与疾病表型共分离，家系未受累者及100名正常人无此突变。因此KIF21A基因为该CFEOM1家系患者的致病基因。6名散发患者存在KIF21A基因的第21外显子的杂合性突变，即c．2860C→T突变。(4)首次报道CFEOM患者合并少年白发。该CFEOM1家系14名患者中有11名患者合并少年白发，家系患者间表现型存在异质性，部分患者存在瞳孔缩小和异常眼球运动。提示突变的KIF21A基因及其蛋白质产物，或者伴随的其他基因的修饰可能干扰了毛发黑色素的代谢。(5)CFEOM1家系和散发患者临床研究、眼外肌病理及眼眶CT检查显示了眼外肌(除下直肌外)存在广泛的发育不良及萎缩，部分患者合并视神经病变，支持CFEOM为原发性神经源性疾病。更多还原

【Abstract】 Objective:Congenital fibrosis of the extraocular muscles (CFEOM) refers to a group ofcongenital eye movement disorders that are characterized by nonprogressiveophthalmoplegia affecting all the extraocular muscles. In this study we described theclinical phenotypes, the pathologic features of the extraocular muscles (EOM) andgenetic features of a Chinese family with autosomal dominant CFEOM and 13sporadic cases with CFEOM and analyzed the genotype-phenotype correlations.1. To clinically characterize a familial and 13 sporadic cases with CFEOM, toinvestigate the pathologic features of EOMs and orbit computerizedtomography (CT) in affected individuals.2. To identify the chromosomal location of disease loci in CFEOM family byLinkage analysis.3. To sequence the candidate gene KIF21A to reveal mutation in this ChineseCFEOM family, direct gene sequence of KIF21A was conducted in 13 sporadiccases with CFEOM to elucidate the genetic background.4. To perform SSCP to conform the mutation state in affected individuals and toexclude it as a polymorphism in a reference population.Methods:1. Clinical study:1.1 Disease histories of patients were recorded. Ophthalmologic examinationincluded corrected visual acuity, refractive error, slit-lamp examination,ophthalmoscopic examination, force of levator palpebrae superioris, eyemovement, ocular position and forced duction testing. 1.2 EOMs biopsy specimens were obtained from CFEOM affected individuals andsubjects underwent enucleation for ocular injury, but none had any ocularmotility disorders. Specimens underwent light and electronic microscopicexamination.1.3 Affected individuals and control subjects correlated with orbit CT studies.2. Molecular genetic study:2.1 Genomic DNA was extracted from 5-8ml peripheral blood leukocytes usingDNA Isolation Kits for Mammalian Blood (Rche Biochimical, Inc).2.2 Polymorphic microsatellite markers at chromosome 12 around the FEOM1locus were selected from the ABI PRISM Linkage Mapping Set-MD10 panelfor linkage analysis. The polymerase chain reactions (PCR) were carried out toamplify each polymorphism. The allele sizes were determined on ABI3130-avant genetic analyses according to an internal size standard and theresults were analyzed using Genescan analysis v3.7 software and Genetyperv3.7 software (Applied Biosystems). Two point analyses for these markerswere calculated using the MILINK program of LINKAGE package (version5.1).2.3 Gene sequence analysisKIF21A mutation analysis was conducted by PCR amplification of exon 8, 20,21 from genomic DNA of all affected members, some unaffected individuals inCFEOM familial and sporadic cases. If KIF21A mutation identified, SSCPwould performed to conform the mutation state.Results:1. Clinical study:1.1 The genetic trait of this pedigree was autosomal dominant inheritance and met toCFEOM1 criterion. There were 49 family members and 14 cases suffering fromCFEOM1 in four generations. They had bilateral congenital blepharoptosis,head-tilt, chin lift and primary gaze fixed in a hypotropic position. But the verticaland horizontal position of eyes and restriction of eye movement were differentamong affected individuals. Some of them also had pupillary abnormally, aberrant innervation and juvenile canities. 11 of 14 affected family members had juvenilecanities before their age of 20, including the deceased one. 11 sporadic cases metto CFEOM1 criteria were born with bilateral ptosis and varying degrees ofophthalmoplegia with both eyes fixed in a hypotropic position. Two individualsmet to CFEOM3 criterion: one case was unilaterally affected, the other with oneeye in orthoptic position and absent ptosis. Some of the affected individualsassociated with aberrant innervation, teeth dysplasia, optic nerve dysplasia andlower eyelid entropion. Inferior rectus recession improved hypotropia in patientswith infraducted eyes and chin elevation. Horizontal muscle recession correctedhorizontal strabismus satisfactorily in most cases. Ptosis was repaired by frontalissling or levator resection.1.2 Microscopic sections taken from medial recti and inferior recti from affectedindividuals contained clumps of myofibers that surrounded by loose connectivetissue. The sizes of myofibers were not even, and some of myofibers were atrophic.Electron microscopy demonstrated replacement of normal muscle by collagen andfibrous tissue in some areas. Vacuolar degenerations and central aggregates ofmitochondria were noted.1.3 All affected individuals examined by CT scanning demonstrated reduction in sizeof the EOMs of affected eyes, particularly the superior rectus and levatorpalpebrae superior, superior obliques, medial recti and lateral recti sometimesbeing affected. The inferior recti were generally normal in size. The optic nervesdisplaced to the superior and nasal part of the orbits and some of them werethinner.2. Genetic molecular study2.1 Linkage analyses were done for the CFEOM family with markers at chromosome12 around the FEOM1 locus. Linkage analysis suggested linkage to markersD12S1048, D12S1663, D12S85, D12S1661, D12S1635 and D12S398. The highestLOD score occurred was 3.41 at D12S1048, suggesting that the disease gene in thefamily may be KIF21A.2.2 Direct DNA sequence analysis from all affected individuals and some unaffected individuals in CFEOM family revealed a heterozygous C→T transition atnucleotide 2860 of KIF21A, which would result in substitution of the Arginineresidue at codon 954 by a Tryptophan residue. The Arg954 residue is highlyconserved during evolution. The p.Arg954Trp mutation was not present inunaffected family members. SSCP analysis confirmed the results of DNAsequencing. Further analysis with SSCP did not detect the mutation in 100 normalcontrols. These results suggested that the p.Arg954Trp substitution of KIF21A isnot a polymorphism, but a causative mutation for CFEOM1 family. 6 sporadiccases also had the heterozygous C→T transition at nucleotide 2860 of KIF21Awhich resulted in p.Arg954Trp substitution.Conclusions:1. We identified a Chinese autosomal dominant inheritance CFEOM1 family with49 members including 14 affected individuals in four generations. We alsoidentified 13 sporadic patients with CFEOM.2. Linkage analysis of the CFEOM1 family was consistent with linkage to thepolymorphic microsatellite markers at chromosome 12 around FEOM1 locus.The maximum LOD score was 3.41 at D12S1048.3. Direct DNA sequence analysis identified a 2860 C→T change in exon 21,resulting in a tryptophan substitution for arginine in codon 954 of KIF21A. SSCPconformed that mutation p.Arg954Trp of KIF21A is the genetic basis of theCFEOM1 family. 6 sporadic cases also had the p.Arg954Trp substitution ofKIF21A.4. This paper is the first we are aware of to describe familial congenital fibrosis ofthe extraocular muscles occurring in association with juvenile canities. 11 of 14affected individuals accompanied with juvenile canities. There was wideintra-familial and inter-ocular phenotypic variability in this CFEOM1 family.Some of the affected members also had pupillary abnormally and aberrantinnervation. It is possible that mutated KIF21A gene and its product with orwithout the influence of a modifying gene interferes with development of cranialnerves, as well as affecting the hair follicle pigmentary unit. 5. The clinical study, muscle pathology and orbit CT scanning in affectedindividuals identified abnormality of optic nerve in some patients, dysplasia andatrophy of EOMs except inferior recti in all affected individuals, supporting thatCFEOM is primary neuropathic process.更多还原