A Chinese Familial And Sporadic Cases With Congenital Fibrosis Of The Extraocular Muscles: Clinical And Molecular Genetic Study

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.