title: Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis.
abstract: BACKGROUND
  Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination.
  
  
  OBJECTIVE
  We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients.
  
  
  METHODS
  We examined 24 Japanese AD patients with "FLG-shotgun" method.
  
  
  RESULTS
  Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients.
  
  
  CONCLUSIONS
  We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.
authors.affiliation: Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sasaki
authors.forename: Jun
authors.initials: J
authors.lastname: Kudoh
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Ebihara
authors.forename: Aiko
authors.initials: A
authors.lastname: Shiohama
authors.forename: Shuichi
authors.initials: S
authors.lastname: Asakawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shimizu
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.forename: Itaru
authors.initials: I
authors.lastname: Dekio
authors.forename: Chieko
authors.initials: C
authors.lastname: Sadahira
authors.forename: Masayuki
authors.initials: M
authors.lastname: Amagai
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D000483:Alleles; D018389:Codon, Nonsense / Q000235:genetics; D004247:DNA / Q000235:genetics*; D003876:Dermatitis, Atopic / Q000097:blood / Q000208:ethnology / Q000235:genetics*; D000091344:Filaggrin Proteins; D005838:Genotype; D006801:Humans; D007381:Intermediate Filament Proteins / Q000097:blood / Q000235:genetics*; D007564:Japan; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D018389:Codon, Nonsense; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D004247:DNA