Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis
Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis
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DOI：
10.1016/j.jdermsci.2008.02.009
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发表时间：
2008-08-01
影响因子：
4.6
通讯作者：
Shimizu, Nobuyoshi
中科院分区：
文献类型：
--
作者：
Sasaki, Takashi;Kudoh, Jun;Shimizu, Nobuyoshi
背景：最近的报道表明，在寻常性鱼鳞病（IV）患者中发现的聚丝蛋白（FLG）无义突变是特应性皮炎（AD）合并哮喘的易感因素。FLG的外显子3包含串联重复、高度同源的11-13个972或975 bp的序列单元，每个单元对应加工后聚丝蛋白的编码序列，序列差异较小。这种独特的基因结构阻碍了DNA序列的精确测定。目的：我们开发了一种新的DNA测序方法“FLG-shotgun”来直接表征日本AD患者的突变。方法：采用“FLG-shotgun”法对24例日本AD患者进行检查。结果：采用多组共同引物对保守区进行PCR扩增，扩增出多个FLG单位，并确定每个克隆PCR产物的DNA序列。两个等位基因的DNA序列的多个读数被对齐和重建，以覆盖整个编码区。我们发现了三种主要的基因型（A、B和C），它们代表不同数量的同源序列单元（11-13）。此外，我们发现了两个新的无义突变；等位基因B第9单元上的一个突变8666-8667CC > GA，导致两名患者无义突变S2899X；等位基因B第10单元上的另一个突变9887C > a，导致两名患者无义突变S3296X。结论：通过对日本AD患者的直接分析，我们发现了两个新的FLG突变。FLG-shotgun将为进一步定义与FLG突变相关的AD表型的性质提供有价值的基础。(C) 2008日本皮肤病研究学会。爱思唯尔爱尔兰有限公司出版。版权所有。
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.Conclusion: We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable toot to further define the nature of the AD phenotype associated with FLG mutations. (C) 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.