應用第三代定序法檢測異位性皮膚炎相關的纖聚蛋白基因突變

Application of Third-generation Sequencing for the Detection of Filaggrin gene Mutations in Atopic Dermatitis

纖聚蛋白是表皮顆粒細胞的主要組成，由filaggrin （FLG）基因所表達，此基因突變與異位性皮膚炎（atopic dermatitis; AD）有關。研究顯示在台灣約有30%的異位性皮膚炎患者帶有FLG基因突變。為探討FLG基因突變之分布，我們共收集9位嚴重異位性皮膚炎患者之DNA，以long range PCR擴增FLG基因第三外顯子片段，並以第三代定序儀PacBio RS II進行基因定序，部份樣本輔以全基因體定序。結果顯示在9位患者中，一位帶有NCBI ClinVar資料庫中已記載的3321delA致病基因突變；另一位找到可能的G10191C及T10186C危險因子。在其他三位病人中找到G5383A、4893_4894del、4896_4897 insA與5003_5010del，可能為新發現的異位性皮膚炎相關基因變異。雖然這些新發現的基因變異與異位性皮膚炎的關聯仍需進一步探討，本研究所建立的FLG基因定序平台與突變資訊，將有助於未來異位性皮膚炎基因檢測的開發。 Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation ofthe skin barrier. Mutations in the gene encoding filaggrin (FLG) have been identified as the cause ofichthyosis vulgaris and have been shown to be major predisposing factors for atopic dermatitis(AD). In addition, FLG mutations are significantly associated with AD-associated asthma andallergic rhinitis. In terminally differentiated keratinocytes, filaggrin is crosslinked to the cornifiedcell envelope, which constitutes an insoluble barrier in the stratum corneum, protecting theorganism against environmental agents and preventing epidermal water loss. The FLG gene resideson chromosome 1q21 and consists of three exons. Exon 3 is extremely large (>12 kb) and encodesmost of the profilaggrin polypeptides with almost completely homologous 10, 11 or 12 repeats.Sequencing of exon 3 is difficult, not only because of its size but also of containing 10 to 12 tandemlyarranged FLG repeat units. According to the epidemiology study of AD from Chang Gangmemorial hospital, the prevalence of AD in children of 6~7 years old is about 10.6%, and 7.4% inchildren of 13~14 years old. Up to date, there is no available molecular diagnostic method for AD inclinic. In this study, we established long range PCR for FLG gene amplification. Long range PCRcan amplify two overlapping PCR fragment for secondary step of ligation to barcode sequences.Two PCR products (6,511 and 5,916 bp) were purified by gel extraction. DNA sequencing will beperformed using Pacific Biosciences RSII DNA sequencer. We found one well-known mutation inAsia population, 3321delA, and other six novel mutations (G10191C, T10186C, G5383A,4893_4894delGA, 4896_4897 insA, and 5003_5010delCATCCCAG) from 9 early onset severe ADpatients. In the future, we will detect and analyze those AD-associated FLG mutations for furtherapplications in clinical laboratory diagnosis by other rapid tests, and establish the allele frequencyof various FLG mutations in Taiwanese.