Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis.
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.
Sasaki
Takashi
T
Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Kudoh
Jun
J
Ebihara
Tamotsu
T
Shiohama
Aiko
A
Asakawa
Shuichi
S
Shimizu
Atsushi
A
Takayanagi
Atsushi
A
Dekio
Itaru
I
Sadahira
Chieko
C
Amagai
Masayuki
M
Shimizu
Nobuyoshi
N
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*
D018389:Codon, Nonsense; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D004247:DNA