Linkage Analysis In Two Chinese Family With Congenital Fibrosis Of Extraocular Muscles And Mutation Screening Of Kif21A

Objective:To describe the clinical phenotypes in two Chinese families with congenital fibrosis of extraocular muscles (CFEOM), and to determine the genetic location of the disease gene by linkage analysis. Elucidate the genetic background of the phenotypes of the two CFEOM families by directly sequencing the candidate gene. Methods:1. Clinical studyDisease histories of patients were recorded. Ophthalmic examination included distance visual acuity, slit-lamp examination, eye movement and ocular position.2. Molecular genetic study2.1 Genomic DNA was extracted from 5-8ml peripheral blood leukocytes using DNA Isolation Kits for Mammalian Blood(Rche Biochimical, Inc).2.2 Linkage analysisLinkage analysis and genome-wide linkage screening were conducted using fluorescent detection of 370 microsatellite markers representing all autosomes at an average resolution of approximately 10 cM (Weberset 6.0). The polymerase chain reactions were carried out to amplify all 370 microsatellite markers. The allele sizes were determined on ABI 377sequencer according to an internal size standard (GeneScan? - 500 TAMRA?, Perkin Elmer, Foster City, USA) and the results were analyzed using Genescan 3.1 and Genotyper 2.0 software (Perkin Elmer). Multipoint Lod scores between the disease status and the marker alleles were calculated using the LINKAGE software package of SimWalk2 2.86, Version 3.35. To refine the candidate region of disease gene, more microsatellite markers at an average resolution of approximately 2 cM were choosed around the location of the microsatellite marker with the largest positive Lod score (part of microsatellite markers were separated by electrophoresis through 6% polyacrylamide gels, Haplotypes were constructed manually according to the pattern of the bands on the gel stained by silver). 2.3 Gene sequence analysisDirect sequence was conducted in all affected members in TS, HL families to determine the mutation in all exons and exon-intron boundaries of KIF21Agene. Results:Two affected members in TS family don't exhibit classic phenotype of CFEOM. Other affected members in this family and all affected members in HL family were born with classic phenotype of CFEOM. The maximum Lod score of 370 microsatellite markers in TS family occurred on D12S1090. Further linkage analysis showed the Markers D12S1648 > D12S345 -D12S1692, D12S59> D12S1090> D12S2194> D12S1048and D12S1668 co-segregated with the disease locus in all affected members in TS family. The maximum Lod Score was 1.91(D12S1090). Recombination events occurred in marker D12S61 and D12S1064, so the candidate region of the disease gene in TS family was located on 12pll.2-ql2. No microsatellite marker with positive...