臺灣博碩士論文加值系統

結節硬化症造成之臉部血管纖維瘤，對病患及家屬雙方皆會造成極大的心理負擔。結節硬化症之致病基因為TSC1及TSC2 ，兩者為控制mTOR分子(mammalian target of rapamycin)的上游蛋白質。Rapamycin為mTOR抑制劑，透過外用藥膏塗抹對結節硬化症引起之臉部血管纖維瘤有一定的成效。Calcitriol為人工合成之維生素D3的衍生物，外用calcitriol可減少鞏皮症的皮膚纖維化，因而對血管纖維瘤可能有治療效果。本研究目的除探討外用單方藥膏rapamycin或calcitriol，或者兩者的合併治療，對結節硬化症造成之臉部血管纖維瘤的療效及安全性外，亦針對皮膚病灶組織進行次世代定序檢測，以期全面找出致病變異點。
本研究方法於皮膚試驗總計納入52位結節硬化症患者，男性20人，女性32人，有50位完成第一階段研究。在研究第一階段，將皮膚病灶分為左右臉兩側，一側使用單方藥膏rapamycin(0.1%)或calcitriol(3mcg/g)，一側使用複方藥膏rapamycin和calcitriol，比較兩者在臉部對稱性血管纖維瘤病灶上療效的差異，為期12週，藥膏的選擇與施用側採隨機分配雙盲方式進行。主要終點評估為各治療組12週臉部血管纖維瘤嚴重度指標(Facial Angiofibroma Severity Index, FASI)和基準期比較的下降程度，FASI為臉部紅斑(erythema)、纖維瘤大小(size)、丘疹隆凸(papule elevation)及臉頰病灶擴展範圍(extension)分級之總分數。在研究第二階段，選用於第12週解盲時較有療效的一側，持續塗抹全臉，進行額外12週的治療(13-24週)。之後停藥12週，於第36週追蹤FASI評估病灶是否復發。次要終點評估為各治療組12週受試者的視覺評估指標(VAS)和基準期比較的下降程度。於皮膚病灶組織納入8位受試者取皮膚Genomic DNA，利用次世代定序及桑格定序確認其致病變異點。
研究結果三個治療組之FASI於12週時與基準期相比皆有達到統計上減少的顯著差異。複方藥膏比起單方藥膏rapamycin或calcitriol能更迅速獲得較佳療效。於12週將單方藥膏calcitriol改為複方藥膏使用的個案，在24週的FASI與12週相比仍具有減少的顯著差異。持續使用複方藥膏或由單方rapamycin改為複方藥膏的個案，在24週與12週相比仍可改善丘疹突出情形，雖然FASI在24週無統計上顯著改善。在24週停藥後持續追蹤至36週，雖然觀察到血管纖維瘤會復發，但36週FASI與基準期比較，使用複方藥膏仍維持統計上減少的顯著差異。三個治療組之受試者VAS於12週時與基準期相比皆有達到統計上減少的顯著差異。而皮膚病灶組織實驗找出兩位為鑲嵌型突變。
外用藥膏無論是單方的rapamycin或calcitriol或是複方rapamycin-calcitriol藥膏，對治療結節硬化症之臉部血管纖維瘤，皆具有療效及安全性，受試者也自覺病灶有顯著改善。而複方藥膏比起單方藥膏更具有顯著且持久的治療效果。利用次世代定序作為基因檢測方式，雖可以全面地找出致病變異點，但對於低比率的鑲嵌型突變及位於非編碼區域變異，仍是一大挑戰。

Tuberous sclerosis complex (TSC)-associated facial angiofibroma is psychologically debilitating to both patients and their family members. The pathogenesis of TSC stems from TSC1 or TSC2 mutations, leading to the defect in mTOR inhibition. Rapamycin is an mTOR inhibitor and is effective for TSC facial angiofibroma through topical administration. Calcitriol, a vitamin D3 analogue, has been shown to lessen skin fibrosis in scleroderma and may be therapeutically beneficial to angiofibromas. The aim of the study is to determine the effect and safety of topical rapamycin or calcitriol and their combination for the treatment of TSC-associated facial angiofibroma. Detected cutaneous lesions using targeted NGS.
A total of 52 TSC patients including 20 male and 32 female subjects were recruited, and 50 of them completed the period 1 study. In period 1, topical rapamycin (0.1%) or calcitriol (3 mcg/g) single-agent therapy versus their combination were applied twice a day by a left-right randomized, split-face comparison for 12 weeks. The primary end point was the reduction of Facial Angiofibroma Severity Index (FASI) for the grade of erythema, papule size, elevation and extension of the lesions at week 12. In period 2, the patients entered an open-label study and were reassigned to use the more effective ointment on both cheeks for another 12 weeks (week 13-24). A follow-up FASI analysis for recurrence after drug discontinue for 12 weeks was also performed (week 36). The secondary end point was the reduction of Visual Analysis Score (VAS) evaluated by the subjects themselves at week 12. A total of 8 patients of cutaneous lesions in TSC were recruited, using NGS identified mutation.
All the three topical agents showed a significant reduction of FASI compared to the baseline at week 12. Rapamycin-calcitriol combination treatment resulted in faster and greater improvements than calcitriol or rapamycin along. The efficacy of combination treatment caused a statistically significant reduction of FASI at week 24 after switching from single calcitriol treatment at week 12. The combination treatment continuously decreased papule elevation after keeping its use for 24 weeks or switching from the single rapamycin treatment at week 12, although the FASI was not significantly improved between week 12 and 24. After discontinuing treatment for 12 weeks, although recurrence was observed, a significant reduction of FASI compared to the baseline still remained. No adverse reactions except transient mild itch on the application site was noted. All the three topical agents showed a significant reduction of VAS compared to the baseline at week 12. No somatic mutation were detected, excepted for two mosaic mutations.
All the three topical agents, which were safe and well-tolerated, showed a significant reduction of FASI and VAS compared to the baseline at week 12. Topical rapamycin-calcitriol combination demonstrated a higher and sustained clinical response compared with the single-agent treatment for TSC-related facial angiofibroma. That are challenges of detecting mosaic mutations and non-coding region even using NGS.

口試委員會審定書 i
致謝 ii
中文摘要 iii
Abstract v
第一章 研究背景與動機 1
1.1. 結節硬化症之疾病介紹 1
1.2. 結節硬化症之歷史演進 2
1.3. 結節硬化症之臨床表徵 3
1.3.1.皮膚表徵 3
1.3.2.神經表徵 4
1.3.3.心臟表徵 4
1.3.4.腎臟表徵 4
1.3.5.肺臟表徵 4
1.3.6.眼部表徵 5
1.3.7.口腔表徵 5
1.3.8.其他器官表徵 5
1.4. 結節硬化症之臨床診斷及檢查 5
1.4.1.基因診斷標準 5
1.4.2.臨床診斷標準 6
1.5. 結節硬化症之治療現況 7
1.6. 結節硬化症基因與Rapamycin、Calcitriol之作用機轉 8
1.7. 遺傳諮詢 9
1.8. 研究動機 10
第二章 研究方法 12
2.1. 研究對象來源及條件 12
2.1.1.受試者來源 12
2.1.2.受試者納入及排除條件 12
2.1.3.受試者退出試驗條件 13
2.1.4.用藥注意事項 13
2.2. 試驗藥物 13
2.2.1.藥物名稱 13
2.2.2.劑型 13
2.2.3.劑量 13
2.2.4.藥物配製 14
2.2.5.藥物與設備 15
2.3. 試驗設計與流程 15
2.4. 療效評估指標 16
2.4.1.主要評估指標 16
2.4.2.次要評估指標 16
2.5. 基因定序檢測 16
2.5.1.皮膚病灶檢體受試者來源 16
2.5.2.皮膚DNA萃取流程 17
2.5.3.次世代定序原理 18
2.5.4.定序結果判讀 19
2.5.5.PCR檢定及Sanger定序判讀 19
第三章 研究結果 20
3.1.人口統計與臨床表徵 20
3.2.療效評估 21
3.2.1.臉部血管纖維瘤嚴重度指標（FASI）改善評估 21
3.2.2.臉部血管纖維瘤紅斑、大小、丘疹及範圍改善評估 22
3.2.3.受試者視覺評估指標（Visual Analysis Score） 26
3.3.外用藥膏副作用評估 27
3.4.基因定序結果統計與分析 28
3.4.1.次世代定序結果統計 28
3.4.2.皮膚病灶檢體之次世代定序結果統計 29
3.5.遺傳諮詢案例 33
3.5.1.案例一 33
3.5.2.案例二 34
3.5.3.案例三 35
第四章 討論 36
第五章 結論 38
參考文獻 39
附錄 56
附錄一、臉部血管纖維瘤外用藥試驗流程圖 56
附錄二、個案評估表 57
附錄三、個案報告表 58
附錄四、病患日誌表 59
附錄五、副作用標準表 60
附錄六、解盲組別對照表 62
附錄七、TSC1及TSC2基因引子序列 63

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