Ultrastructural Study Of The Extraocular Muscles And Analysis Of Gene Mutation In Congenital Nystagmus

ObjectiveTo treat congenital nystagmus(CN)by modified Kestenbaum procedure for improving compensatory head posture and visual acuity in primary position.To observe the ultrastructural changes of the extraocular muscles(EOMs)in CN subjects and discuss the probable effect and its mechanism of these alterations on CN.To identify the genetic location of the candidate gene in two Chinese families with X-linked congenital nystagmus.To analyze the sequence of candidate gene FRMD7 by directly sequencing to reveal mutation and study the molecular pathogenesis of the CN in Chinese family.Methods1.Modified Kestenbaum procedure1.1 Routine ophthalmologic examinations including best corrected visual acuity, refractive error,ocular anterior segment,fundus,ocular position,eye movement, direction and degree of compensatory head posture,prism adaption test.1.2 Modified Kestenbaum procedure was conducted on patients with CN. When the angle of compensatory head posture was from 25°to 30°,Parks 5-6-7-8 method was choosen.Modified Parks method was carried out if the angle of compensatory head posture was more than 30°.2.Ultrastructural study of EOMsTo observe the morphological changes of the removed EOMs with light and transmission electron microscopy.Controls were obtained from subjects underwent enucleation for ocular injury,but none had any ocular motility disorders.3.Molecular genetic study3.1 Human Genomic DNA was extracted from 5-8ml peripheral blood leukocytes using DNA Isolation Kits for Mammalian Blood(Rche Biochimical,Inc).3.2 Polymorphic microsatellite marks at Xq25-q27 were selected.Fluorescently labeled microsatellite markers were analyzed by polymerase chain reaction(PCR) amplification.The allele sizes were determined on ABI3130-avant Genetic Analyzer and the results were analyzed using GeneMapper 3.7 software.Two point analysis for these markers were calculated using the MILINK program of LINKAGE package (version5.1).3.3 Gene sequence analysisDirect genomic sequencing was used to evaluate all the exons of FRMD7 gene. FRMD7 mutation analysis was conducted on all affected members in two families. Once the probable mutation was identified,120 unaffected individuals were screened to exclude the mutation as a polymorphism in a reference population.Results1.Modified Kestenbaum procedure:compensatory head posture of all 5 patients has disappeared,visual acuity in primary position averagely increased 0.32 lines, tropia wasn't found among patients after surgery.2.Ultrastructural study of EOMs:myofibrillae of the EOMs arranged irregularly, parallel fibre became confused and intercrossed.Some myofibrillae were fragmented and dissolved,but collagen fibre proliferated.Mitochondria was swollen in which the inner crest was arranged disorderly even collapsed and disappeared.The proprioceptors in the EOMs of CN subjects were damaged and their external capsules became collapsed,discontinuous with inhomogeneous electron-dense.The myelin of myeiinated nerve endings exhibited degeneration and dissociation.Mitochondria was swollen and vacuolated.Myeloid body was found in axon.When the abnormality was severe,Schwann's cells were necrotic,components of axon were dissolved and disappeared in capsules.3.Molecular genetic study:the disease-causing gene was closely linked to DXS 1047 in two Chinese families.Haplotype construction of the family HU defined the disease interval between DXS8059 and DXSS033.The candidate region of disease gene of the family LI was defined between DXS8044 and DXS1227.The disease locus of two families were overlapped,this results narrowed the CN locus down to about a 4cM region between DXS8044 and DXS8033 at chromosome Xq25-Xq26.3.Direct DNA sequence analysis revealed a heterozygous mutation in exon9 and a 2bp deletion in exon8 of the FRMD7 gene in all affected member of two families respectively. Conclusion1.Modified Kestenbaum procedure is safe and effective to improve compensatory head posture and visual acuity in primary position of CN patients.2.The ultrastructure of the muscle fiber and proprioceptors in the EOMs of CN subjects are obviously changed compared with the controls.These alterations probably related to the etiology and pathogenesis of CN.3.The candidate region of disease gene of two Chinese families with X-linked congenital nystagmus was located on Xq25-Xq26.3,defined between DXS8044 and DXS8033.4.Two novel mutations of the FRMD7 gene:812G＞T and 689-690delAG were reported firstly.