結論 - 結節硬化症：REDCap資料庫的建立以及次世代定序基因檢測

本研究整理了臺大醫院結節硬化症整合門診的病人 508 位，共 222 個家族，

包含家族史、臨床表徵、基因檢測、各科追蹤檢查紀錄等資訊，在臺大安全網域下建構線上 TSC REDCap 資料庫。在本研究收納的臺灣結節硬化症病人中，次世代定序基因檢測確診檢出率約 88%，3%為 VUS，9%沒有發現任何懷疑的變異 點。其中有 20%為 TSC1 基因變異，80%為 TSC2 基因變異；30%為遺傳而來，

70%為新發生的變異。臺灣本土結節硬化症流行病學狀況與過去國外研究沒有明顯的差異[8]。

利用分析兩代直系親屬基因，可確認一同時患有結節硬化症和多囊腎的病人身上兩個致病變異點在染色體上的相位關係；從過去基因檢測未找到致病變異點位的病人之中，利用皮膚病灶檢體可以提高低比例鑲嵌型的患者，生殖細胞突變 被檢測出來的比率；帶有 TSC2 生殖細胞突變且同時為 B 型肝炎帶原的結節硬化 症病人若患有 HCC，則其 HCC 腫瘤會較大且侵略性更強。在其 HCC 腫瘤切片細 胞發現除了患者原本的一個 TSC2 生殖細胞突變之外，另有一段約 2Mb 包含

TSC2 區域的缺失，使此區域由原本的異型合子變為 LOH 的狀況。使用免疫治療

搭配 everolimus 治療對控制此病人的腫瘤擴張是有效果的，和過去文獻一致[52, 54]；最後，利用 minigene 來確認未確定致病性的剪接位變異點其轉錄出的 mRNA 是否有異，目前實驗仍在持續進行中。

第六章參考文獻

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附錄一、PCR primers

(1) TSC1 PCR primers

Exon No. name forward reverse size (bp) Tm

3 TSC1-3F acacgtgcattagtttgtct

483 50 TSC1-3R taagaatcatgcagtgaacc ggttcactgcatgattctta

4 TSC1-4F gaactgtaatgctgcacaaa

418 50 TSC1-4R gtaggacccatgattcttga tcaagaatcatgggtcctac

5 TSC1-5F tgacccttgctttacattag

429 50 TSC1-5R aatgttgtccagagttggta taccaactctggacaacatt

6 TSC1-6F aatccttagggatagggaaa

463 50 TSC1-6R tccactacaggcacatagaa ttctatgtgcctgtaggtgga

7 TSC1-7F tttggagtgaggtgggaata

368 50 TSC1-7R tatttaacggcagacaggga tccctgtctgccgttaaata

8 TSC1-8F gtgactggcttctttcactc

371 58 TSC1-8R cacttggaagcactcataca tgtatgagtgcttccaagtg

9 TSC1-9F gcactgagttgacactctga

398 50 TSC1-9R cttaaatttgcatgtccctc gagggacatgcaaatttaag

10 TSC1-10F ccagaataacctaaaaccac

340 50 TSC1-10R ctgttgcctcttagattcat atgaatctaagaggcaacag

11 TSC1-11F cactggccatggatataaac

320 50 TSC1-11R acacttattgcaaatccctg cagggatttgcaataagtgt

12 TSC1-12F ccagaaagttaactctagca

322 50 TSC1-12R ttgagaagcctaattatgcc ggcataattaggcttctcaa

13 TSC1-13F catcccaacaatttgagaat

308 50 TSC1-13R tccaaaatactgaggcctaa ttaggcctcagtattttgga

14 TSC1-14F ttgtagtatccccaggacag

324 50 TSC1-14R gacctatgccaggtaaagtg cactttacctggcataggtc

15 TSC1-15F tgccactttttctcctctc

780 60 TSC1-15R atcattcttcacactcccac gtgggagtgtgaagaatgat

16 TSC1-16F ctgcaaataaaagtccaga

219 50 TSC1-16R cctctgggaaacagatctc gagatctgtttcccagagg

17 TSC1-17F agctaacaacacatgggaag

390 50 TSC1-17R caggtcgcttgagattaaga tcttaatctcaagcgacctg

TSC1-18F aatgttcgcagtgtgtgtta

383

TSC1-18R gctgaatgcttttctctctg cagagagaaaagcattcagc

19 TSC1-19F aaagtagagccgttgagcta

331 50 TSC1-19R cttgagtgtctgtgtcatgg ccatgacacagacactcaag

20 TSC1-20F cctagctgattccctgttta

321 50 TSC1-20R tatttcctaaaagacggagc gctccgtcttttaggaaata

21 & 22 TSC1- 2122F tgccttctcagtccttctta

638 50 TSC1-2122R actagacaggctgcaacact agtgttgcagcctgtctagt

23 TSC1-23F tagcggagttcagtgtcagt

745 50 TSC1-23R caagttcatgtgtggaaatg catttccacacatgaacttg

使用 GoTaq® DNA Polymerase system

(2) TSC1, PKD1 PCR primers

Exon No. name forward reverse size Tm

1 TSC2-1F-1 gctttcctaggtgcctgtttg

548 60 TSC2-1R-1 gacctggttcaggcacactt aagtgtgcctgaaccaggtc

2 TSC2-2F-1 tcaagtgaatcttgattccagaaag

738 60 TSC2-2R-1 caatcaagcgtggaaatgggg ccccatttccacgcttgattg

3 TSC2-3F gttgttcctccctgtcc

333 60 TSC2-3R acagggtctaggggctga tcagcccctagaccctgt

4 TSC2-4F ctctgtgggaaggagagg

398 60 TSC2-4R gtatgcccaccctgcttc gaagcagggtgggcatac

5 TSC2-5F-1 caggagtctggtgatgtcgg

508 60 TSC2-5R-1 gctccgcagtgaataaagttgaa ttcaactttattcactgcggagc

6 TSC2-6F atgagccatgcgtgttat

280 53 TSC2-6R aagctgctcggtctctct agagagaccgagcagctt

7 TSC2-7F-1 tcaatgacccacagtgacagg

538 62 TSC2-7R-1 gtctttgggaggagatggtgg ccaccatctcctcccaaagac

8 TSC2-8F-1 gtgtcattttcccaggcagt

417 60-65 TSC2-8R-1 gttccagaggctgccactag ctagtggcagcctctggaac

9 TSC2-9F gcaacctcacacatccat

322 55 TSC2-9R catttctaggcctttcca tggaaaggcctagaaatg

10 TSC2-10F ctctcggtcccaagg

369 55 TSC2-10R tctctggtgattcgcagt actgcgaatcaccagaga

11 TSC2-11F ctgagggtgtctccatgc

369 58 TSC2-11R ctttccaggtttctgcac gtgcagaaacctggaaag

12 TSC2-12F-1 caggcttttccatgggtttgg

459 62

TSC2-12R-1 tagcttgctttccagtccagc gctggactggaaagcaagcta

Exon No. name forward reverse size Tm 13 TSC2-13F-1 ggtctcaggatgcccgatg

536 60 TSC2-13R-1 cgtggtctgtttacccctgt acaggggtaaacagaccacg

14 TSC2-14F cctgaggaattggaagtgtc

351 55 TSC2-14R ggctgtgtccgtaggtgagg cctcacctacggacacagcc

15 TSC2-15F-1 ttctgcacagtcactcgggta

981 66

TSC2-15R-1 caccctccctggatttgctg cagcaaatccagggagggtg 16 TSC2-16F-1 gggtttgaaggtcgtgtgtt

468 60 TSC2-16R-1 tctgtggcttgtcgctctaa ttagagcgacaagccacaga

17&18 TSC2-1718F-1 atcagcaggtggccttttctg

675 60 TSC2-1718R-1 tccctcagagctgagccttc gaaggctcagctctgaggga

19 TSC2-19F-1 tagcttccgcctctgtctcta

506 62 TSC2-19R-1 tgtgttacttggcaggcactc gagtgcctgccaagtaacaca

20 TSC2-20F actcccaccactccgaaagg

380 60 TSC2-20R cccaggtaggccccacattt aaatgtggggcctacctggg

21 TSC2-21F gcattcagggacttgcta

403 60 TSC2-21R cctctctcaggactccttg caaggagtcctgagagagg

21-1 TSC2-21-1F aggcattcagggacttgcta

543 60 TSC2-21-2R gtgaacgctcctccttgaag cttcaaggaggagcgttcac

22&23 TSC2-2223F ttctctcctctgcagcac

576 55 TSC2-2223R cagagtgacaggcaggtg cacctgcctgtcactctg

24 TSC2-24F ctagcctgcagcttgtcc

297 56 TSC2-24R aagtcagtgagtggaaat atttccactcactgactt

25 TSC2-25F gggatctctccatcctga

344 55 TSC2-25R ctgcttgcatgacctcat atgaggtcatgcaagcag

26-28 TSC2-2628F tttggcccttggtgatag

782 60 TSC2-2628R ctgctggtcccagtgttc gaacactgggaccagcag

29 TSC2-29F-1 gttcactgaggccagcact

486 60 TSC2-29R-1 cccccaaatatcccaagaggg cccccaaatatcccaagaggg

30 TSC2-30F-1 gtgcaaagagtaggggttcca

690 60 TSC2-30R-1 gtctgccctgctcaggaa ttcctgagcagggcagac

31 TSC2-31F cctgccctctctcctctg

239 55 TSC2-31R ataggtgctgtgctcgtc gacgagcacagcacctat

32 TSC2-32F-1 tgtccctctggtcaggagaag

445 60 TSC2-32R-1 gggatgctgatacctctgctc gagcagaggtatcagcatccc

33 TSC2-33F caccacctccaggtcaac 730 60

Exon No. name forward reverse size Tm TSC2-33R tggccaggctctgtgt acacagagcctggcca

34&35 TSC2-3435F tctgtgttcctccctgtg

556 60 TSC2-3435R acagagctcaacactgcc ggcagtgttgagctctgt

36 TSC2-36F ggaatggatggtcttgtctg

392 60 TSC2-36R ccatccagtcctgctacccc ggggtagcaggactggatgg

37 TSC2-37F cagagcccctggagtaat

350 55 TSC2-37R aggagcaggaggagaggc gcctctcctcctgctcct

38 TSC2-38F aaagttcaggggcagatgct

362 60 TSC2-38R agatcgtgtctgaccgcaac gttgcggtcagacacgatct

38&41 TSC2-3841F-1 gctgacaggtgtctagcagtg

983 60 TSC2-3841R-1 ttgactttgtctgcttggtgc gcaccaagcagacaaagtcaa

1-16 del TSC2-320-del-2F tcttagcctggatggtcttgat

418 63 TSC2-320-del-2R caaagcctcacagcatcactt aagtgatgctgtgaggctttg

1-del TSC2-057-del-1F gctccgagcatcccttagttt

190 60 TSC2-057-del-1R ctacctctgcctcccagtttc gaaactgggaggcagaggtag

6-16 dup TSC2-224-dup-1F tcactcgggtataaagggcat

574 60 TSC2-224-dup-1R aatgttgcatgagctctgtctca tgagacagagctcatgcaacatt

whole

15 TSC338-PKD1-ex15-F ccgtcaccttctacccgcaccc

365 60 TSC338-PKD1-ex15-R cacatgctccactgttgcctcc

使用 GoTaq® DNA Polymerase system

(3) Minigene PCR primers for insert DNA

ggagacccaagctgggcctttgacttcctgttgct 884 bp AccuPrime Taq

ggagacccaagctgggccatggcatgtccgaacga 1961 bp PrimeSTAR GXL

ggagacccaagctggggccagttcaactttgtccacg 493 bp AccuPrime Taq

ggagacccaagctggcgttttcttgcttggggcgaaa 3052 bp PrimeSTAR GXL

ggagacccaagctgggccacgtggcggaagtctat 9722 bp TaKaRa LA Taq

TSC160-TSC1-ex7-9-DNA-R

gccctctagactcgacatagctattctgtgtgtcagc

(4) PCR programs

1. GoTaq® DNA Polymerase system

GoTaq® DNA Polymerase 12.5μl 95

°C, 5 min

95

°C, 30 sec

2.AccuPrime Taq DNA Polymerase system

3. PrimeSTAR GXL DNA Polymerase system PrimeSTAR GXL DNA

4. TaKaRa LA Taq DNA Polymerase system

TaKaRa LA Taq (5U/

μL) 0.5μl

95

°C, 1 min

(5) minigene primers for Sanger sequencing

case primer sequence

TSC144 1 TSC144-TSC2-ex18-R ggatgtcccagggttgggaa

2 1718R-1 gaaggctcagctctgaggga

3 19F-1 tagcttccgcctctgtctcta

4 TSC144-TSC2-ex18-20-DNA-R

gccctctagactcgacatggagcagagagcagagc

TSC160 1 TSC160-TSC1-ex7-9-DNA-F ggagacccaagctgggccacgtggcggaagtctat

2 TSC1-7R tccctgtctgccgttaaata

3 TSC1-8F gtgactggcttctttcactc

4 TSC160-TSC1-in8-F1 ccagttgactataccatcct 5 TSC160-TSC1-in8-F2 agctagttgactaggagg 6 TSC160-TSC1-in8-F3 gctgtcatcctagtctcct 7 TSC160-TSC1-in8-F4 ctgttgttacagttactgc 8 TSC160-TSC1-in8-F5 ctatagagaataatccaggt 9 TSC160-TSC1-in8-F6 gcatatgtaagcatcaggatgc 10 TSC160-TSC1-in8-F7 ggattgaaggatatgagt 11 TSC160-TSC1-in8-F8 catagtatactctgtggct 12 TSC160-TSC1-in8-F9 ctagtcatgctgagaatgc 13 TSC160-TSC1-in8-F10 gctcaagtgatcctcctgct 14 TSC160-TSC1-in8-F11 gctgagattacaggtacgagc 15 TSC160-TSC1-in8-F12 gcagtgagtcgagattgc 16 TSC160-TSC1-in8-F13 gcagatcgatagttgcaag

17 TSC1-9F gcactgagttgacactctga

18 TSC160-TSC1-ex7-9-DNA-R gccctctagactcgacatagctattctgtgtgtcagc TSC201 1 TSC201-TSC2-ex1-R1 agcgcgaggatgctgacc

2 TSC201-TSC2-ex1-R2 atgctcggagctccggag 3 TSC201-TSC2-in1-F1 ggtaagtggcggtcc 4 TSC201-TSC2-in1-F2 gtcttgcaccaggttctgt 5 TSC201-TSC2-in1-F3 ggctggtcttgaactgct

6 TSC2-1R-1 aagtgtgcctgaaccaggtc

7 TSC2-2F-1 tcaagtgaatcttgattccagaaag 8 TSC201-TSC2-ex1-3-DNA-R1 gccctctagactcgactcttcaaatttcttgg

tttttgcgacttcacaaatctgccc TSC203 1

TSC203-TSC2-ex11-13-DNA-F

ggagacccaagctgggccatggcatgtccgaacga

2

TSC2-10R

actgcgaatcaccagaga 3

TSC203-TSC2-in11-F

gctagtatccagcagcct 4

TSC2-11F

ctgagggtgtctccatgc 5

TSC2-12F-1

caggcttttccatgggtttgg TSC270 1

TSC270-TSC2-ex39-41-DNA-F

ggagacccaagctggggccagttcaactttgtccacg

2

TSC270-TSC2-ex39-41-DNA-R

gccctctagactcgaccgctggcggagccgcttgat

附錄三、minigene assay 實驗流程

1. 準備 linearized vector 和 insert DNA

利用 pcDNA3.1，為約 5.4kb 的載體 (vector)，上面已設計了多種限制酶切位，本實驗利用的是其中的Nhe1 和 Xho1 兩個限制酶切位。先用 Nhe1 和 Xho1 限制酶將載體 (vector) 切開成線狀。

使用 NA12878 標準品之 genomic DNA，在待測上下游剪接位變異點旁之外 clone 入 pcDNA3.1 的

insert DNA。在設計 PCR 引子的時候，引子兩邊會額外帶有一段 15bp 和

pcDNA3.1 要 clone 入 insert DNA 的接點兩旁周圍互補的序列，以備之後 cloning 的進行。PCR 出的產物以 PCR clean up and Gel Extraction kit from Omics Bio 進行純化。

2. 將 insert DNA clone 入 linearized vector

利用 Fusion® HD Cloning Kit (Takara)，進行 Gibson assembly。將 5X In-Fusion HD Enzyme Premix、linearized vectors (100 ng, <10 kb)、PCR-generated sequences (100 ng, 0.5 to 10 kb )、ddH2O 混合成 10μl，在 50 °C 乾浴槽作用 15 分鐘，置於冰上作用 5 分鐘，混勻。

將產物分為兩組，其中一組利用Q5 Site-Directed Mutagenesis Kit接著進行 mutagenesis，製造出欲測試的變異點位。

3.transformation to DH5α E. coli competent cells

將 10 μL Gibson assembly reaction mixture 加入 DH5α E. coli competent Cell，

置於冰上30 分鐘，於 42°C 水浴 45 秒進行 heat shock 將 vector 吞入細胞，再置於冰上5 分鐘。

加入 1 cc. S.O.C medium，置於 30°C 下 30 分鐘 225 rpm，促使細菌開始表現抗藥性基因。取100μL ~ 120μL 菌液至 LB plate 塗盤，在 30°C 下培養至隔夜。

4.colony PCR 確認 insert DNA 是否接入 vector

挑選數顆 colony，用 tip 沾取部分菌，於裝有 10.5μl ddH20 的 eppendorf 中混和均勻，再加入針對目標外顯子的PCR 引子： 1μl primer-F 和 1μl primer-R，

以及12.5μl GoTaq ® Green Master Mix，進行 PCR，並電泳確認產物大小正確，

表示insert DNA 有接入 vector。確認有接入 vector 的菌落，挑菌種在 5cc LB broth，並添加額外 15μl ampicillin (150μg/ml)，於 30°C 225 rpm 培養至隔夜。

PCR program: 94°C 3’ buffer，混和均勻，移入 1.5ml eppendorf；加入 250μl MXB2，輕輕上下翻轉 4~6 次，靜置室溫5min；加入 350μl MXB3 中和 lysate，輕輕上下翻轉 4~6 次直至白色沉澱物出現。離心13000rpm 10min，上清液移置 column 中，離心 9000rpm 60 sec；加入 500μl WE buffer，離心 9000rpm 60sec，丟棄過濾液；加入 700μl WS buffer，離心 9000rpm 60sec，丟棄過濾液體；離心 13000rpm 3min，去除酒精，

將column 移置 1.5ml eppendorf；加 50μl ddH2O 至 membrane 正中央回溶，靜置3 min，離心 13000 rpm 3min，測 O.D，-20°C 保存。取部分以限制酶 Nhe1 和Xho1 切割，電泳跑膠，送桑格定序，確認序列無誤。

6.transfection to HEK293

使用 Lipofectamine 3000 Reagent kit，取 8.25μl Lipofectamine™ 3000 Reagent

於1.5ml eppendorf，並以 250μl Opti-MEM™ (Reduced Serum Medium)稀釋後混勻；取 5.5 μg DNA 於 250μl Opti-MEM™稀釋於 1.5ml eppendorf，加入 11 μl P3000™ Reagent 混勻。再將以上兩管相加混和均勻(DNA-lipid complex)，室溫放置10 min。

將細胞株 HEK293 (Human embryonic kidney cells 293)，以 PBS wash 乾淨，

每盤加入4 CC serum free medium。再加入上述 DNA-lipid complex，培養 24hr 後換成含有serum 的 medium。於培養 48hr 抽 RNA。萃取 RNA 後進行 RT-PCR，

最後送桑格定序，觀察序列剪接狀況。

附錄三、TSC panel

chr NM_number_gene name start end length chr NM_number_gene name start end length

chr9 NM_000368_TSC1 135746734 135870020 123286 chr6 NM_138694_PKHD1_exon_67 51480094 51484368 4274 chr16 NM_000548_TSC2 2047895 2158721 110826 chr6 NM_138694_PKHD1_exon_66 51491744 51491964 220 chr16 NM_000296_PKD1 2138710 2195899 57189 chr6 NM_138694_PKHD1_exon_65 51497312 51497571 259 chr17 NM_144606_FLCN 17105526 17160502 54976 chr6 NM_138694_PKHD1_exon_64 51503596 51503804 208 chr1 NM_004958_MTOR_exon_58 11166537 11167607 1070 chr6 NM_138694_PKHD1_exon_63 51512778 51512966 188 chr1 NM_004958_MTOR_exon_57 11168187 11168393 206 chr6 NM_138694_PKHD1_exon_62 51513832 51514068 236 chr1 NM_004958_MTOR_exon_56 11169296 11169477 181 chr6 NM_138694_PKHD1_exon_61 51523699 51524817 1118 chr1 NM_004958_MTOR_exon_55 11169655 11169836 181 chr6 NM_138694_PKHD1_exon_60 51609132 51609390 258 chr1 NM_004958_MTOR_exon_54 11172858 11173024 166 chr6 NM_138694_PKHD1_exon_59 51611468 51611737 269 chr1 NM_004958_MTOR_exon_53 11174324 11174560 236 chr6 NM_138694_PKHD1_exon_58 51612534 51613513 979 chr1 NM_004958_MTOR_exon_52 11174819 11174994 175 chr6 NM_138694_PKHD1_exon_57 51617948 51618201 253 chr1 NM_004958_MTOR_exon_51 11175402 11175575 173 chr6 NM_138694_PKHD1_exon_56 51619531 51619786 255 chr1 NM_004958_MTOR_exon_50 11177010 11177193 183 chr6 NM_138694_PKHD1_exon_55 51637449 51637637 188 chr1 NM_004958_MTOR_exon_49 11181252 11181475 223 chr6 NM_138694_PKHD1_exon_54 51640555 51640769 214 chr1 NM_004958_MTOR_exon_48 11181985 11182233 248 chr6 NM_138694_PKHD1_exon_53 51655983 51656221 238 chr1 NM_004958_MTOR_exon_47 11184504 11184740 236 chr6 NM_138694_PKHD1_exon_52 51695608 51695837 229 chr1 NM_004958_MTOR_exon_46 11186628 11186903 275 chr6 NM_138694_PKHD1_exon_51 51701151 51701317 166 chr1 NM_004958_MTOR_exon_45 11187016 11187251 235 chr6 NM_138694_PKHD1_exon_50 51712522 51712818 296 chr1 NM_004958_MTOR_exon_44 11187630 11187913 283 chr6 NM_138694_PKHD1_exon_49 51720640 51720918 278 chr1 NM_004958_MTOR_exon_43 11188010 11188233 223 chr6 NM_138694_PKHD1_exon_48 51732610 51732957 347

chr1 NM_004958_MTOR_exon_42 11188460 11188659 199 chr6 NM_138694_PKHD1_exon_47 51735251 51735487 236 chr1 NM_004958_MTOR_exon_41 11188861 11189058 197 chr6 NM_138694_PKHD1_exon_46 51747840 51748075 235 chr1 NM_004958_MTOR_exon_40 11189744 11189945 201 chr6 NM_138694_PKHD1_exon_45 51750614 51750820 206 chr1 NM_004958_MTOR_exon_39 11190535 11190884 349 chr6 NM_138694_PKHD1_exon_44 51751880 51752093 213 chr1 NM_004958_MTOR_exon_38 11193086 11193304 218 chr6 NM_138694_PKHD1_exon_43 51768344 51768575 231 chr1 NM_004958_MTOR_exon_37 11194357 11194573 216 chr6 NM_138694_PKHD1_exon_42 51768733 51768890 157 chr1 NM_004958_MTOR_exon_36 11199310 11199542 232 chr6 NM_138694_PKHD1_exon_41 51770962 51771188 226 chr1 NM_004958_MTOR_exon_35 11199539 11199765 226 chr6 NM_138694_PKHD1_exon_40 51774030 51774322 292 chr1 NM_004958_MTOR_exon_34 11204654 11204862 208 chr6 NM_138694_PKHD1_exon_39 51776546 51776804 258 chr1 NM_004958_MTOR_exon_33 11204974 11205152 178 chr6 NM_138694_PKHD1_exon_38 51777113 51777424 311 chr1 NM_004958_MTOR_exon_32 11206682 11206898 216 chr6 NM_138694_PKHD1_exon_37 51798857 51799170 313 chr1 NM_004958_MTOR_exon_31 11210132 11210333 201 chr6 NM_138694_PKHD1_exon_36 51824617 51824874 257 chr1 NM_004958_MTOR_exon_30 11217158 11217398 240 chr6 NM_138694_PKHD1_exon_35 51875056 51875307 251 chr1 NM_004958_MTOR_exon_29 11227448 11227624 176 chr6 NM_138694_PKHD1_exon_34 51882157 51882477 320 chr1 NM_004958_MTOR_exon_28 11259264 11259510 246 chr6 NM_138694_PKHD1_exon_33 51887548 51887792 244 chr1 NM_004958_MTOR_exon_27 11259547 11259810 263 chr6 NM_138694_PKHD1_exon_32 51889321 51891029 1708 chr1 NM_004958_MTOR_exon_26 11264567 11264810 243 chr6 NM_138694_PKHD1_exon_31 51892576 51892744 168 chr1 NM_004958_MTOR_exon_25 11269318 11269565 247 chr6 NM_138694_PKHD1_exon_30 51892903 51893199 296 chr1 NM_004958_MTOR_exon_24 11270820 11271013 193 chr6 NM_138694_PKHD1_exon_29 51897777 51898013 236 chr1 NM_004958_MTOR_exon_23 11272318 11272581 263 chr6 NM_138694_PKHD1_exon_28 51900338 51900569 231 chr1 NM_004958_MTOR_exon_22 11272802 11273015 213 chr6 NM_138694_PKHD1_exon_27 51907606 51907982 376 chr1 NM_004958_MTOR_exon_21 11273405 11273673 268 chr6 NM_138694_PKHD1_exon_26 51908372 51908578 206

chr1 NM_004958_MTOR_exon_20 11276154 11276341 187 chr6 NM_138694_PKHD1_exon_25 51909713 51909936 223 chr1 NM_004958_MTOR_exon_19 11288674 11289025 351 chr6 NM_138694_PKHD1_exon_24 51910751 51911036 285 chr1 NM_004958_MTOR_exon_18 11290931 11291161 230 chr6 NM_138694_PKHD1_exon_23 51913239 51913467 228 chr1 NM_004958_MTOR_exon_17 11291306 11291541 235 chr6 NM_138694_PKHD1_exon_22 51914904 51915143 239 chr1 NM_004958_MTOR_exon_16 11292442 11292635 193 chr6 NM_138694_PKHD1_exon_21 51917823 51918099 276 chr1 NM_004958_MTOR_exon_15 11293404 11293594 190 chr6 NM_138694_PKHD1_exon_20 51918785 51919013 228 chr1 NM_004958_MTOR_exon_14 11294149 11294372 223 chr6 NM_138694_PKHD1_exon_19 51920334 51920577 243 chr1 NM_004958_MTOR_exon_13 11297849 11298155 306 chr6 NM_138694_PKHD1_exon_18 51921445 51921636 191 chr1 NM_004958_MTOR_exon_12 11298408 11298724 316 chr6 NM_138694_PKHD1_exon_17 51921637 51921827 190 chr1 NM_004958_MTOR_exon_11 11300309 11300654 345 chr6 NM_138694_PKHD1_exon_16 51923070 51923449 379 chr1 NM_004958_MTOR_exon_10 11301559 11301788 229 chr6 NM_138694_PKHD1_exon_15 51924675 51924890 215 chr1 NM_004958_MTOR_exon_9 11303120 11303407 287 chr6 NM_138694_PKHD1_exon_14 51927266 51927508 242 chr1 NM_004958_MTOR_exon_8 11307631 11307840 209 chr6 NM_138694_PKHD1_exon_13 51929702 51929898 196 chr1 NM_004958_MTOR_exon_7 11307825 11308201 376 chr6 NM_138694_PKHD1_exon_12 51930723 51930925 202 chr1 NM_004958_MTOR_exon_6 11313845 11314080 235 chr6 NM_138694_PKHD1_exon_11 51934204 51934375 171 chr1 NM_004958_MTOR_exon_5 11315998 11316299 301 chr6 NM_138694_PKHD1_exon_10 51935153 51935293 140 chr1 NM_004958_MTOR_exon_4 11316939 11317272 333 chr6 NM_138694_PKHD1_exon_9 51935753 51935918 165 chr1 NM_004958_MTOR_exon_3 11318491 11318700 209 chr6 NM_138694_PKHD1_exon_8 51936862 51937037 175 chr1 NM_004958_MTOR_exon_2 11319254 11319530 276 chr6 NM_138694_PKHD1_exon_7 51938210 51938389 179 chr1 NM_004958_MTOR_exon_1 11322451 11322658 207 chr6 NM_138694_PKHD1_exon_6 51941023 51941181 158 chr1 NM_006252_PRKAA2_exon_1 57110939 57111204 265 chr6 NM_138694_PKHD1_exon_5 51944647 51944856 209 chr1 NM_006252_PRKAA2_exon_2 57140003 57140245 242 chr6 NM_138694_PKHD1_exon_4 51947139 51947390 251

chr1 NM_006252_PRKAA2_exon_3 57157016 57157210 194 chr6 NM_138694_PKHD1_exon_3 51947925 51948103 178 chr1 NM_006252_PRKAA2_exon_4 57157980 57158225 245 chr6 NM_138694_PKHD1_exon_2 51949629 51949865 236 chr1 NM_006252_PRKAA2_exon_5 57159387 57159575 188 chr6 NM_138694_PKHD1_exon_1 51952181 51952473 292 chr1 NM_006252_PRKAA2_exon_6 57161557 57161882 325 chr8 NM_001283012_DEPTOR_exon_1 120885844 120886258 414 chr1 NM_006252_PRKAA2_exon_7 57169593 57170198 605 chr8 NM_001283012_DEPTOR_exon_2 120977421 120977700 279 chr1 NM_006252_PRKAA2_exon_8 57171714 57171941 227 chr8 NM_001283012_DEPTOR_exon_3 121013713 121013999 286 chr1 NM_006252_PRKAA2_exon_9 57173097 57181058 7961 chr8 NM_001283012_DEPTOR_exon_4 121015165 121015400 235 chr2 NM_005544_IRS1_exon_2 227595982 227601016 5034 chr8 NM_001283012_DEPTOR_exon_5 121018993 121019164 171 chr2 NM_005544_IRS1_exon_1 227659654 227663556 3902 chr8 NM_001283012_DEPTOR_exon_6 121021217 121021422 205 chr3 NM_001130081_PLD1_exon_26 171318144 171321142 2998 chr8 NM_001283012_DEPTOR_exon_7 121061764 121063207 1443 chr3 NM_001130081_PLD1_exon_25 171323038 171323256 218 chr9 NM_001006617_MAPKAP1_exon_12 128199622 128201341 1719 chr3 NM_001130081_PLD1_exon_24 171330018 171330272 254 chr9 NM_001006617_MAPKAP1_exon_11 128206729 128206927 198 chr3 NM_001130081_PLD1_exon_23 171338125 171338360 235 chr9 NM_001006617_MAPKAP1_exon_10 128230200 128230438 238 chr3 NM_001130081_PLD1_exon_22 171360579 171360729 150 chr9 NM_001006617_MAPKAP1_exon_9 128246671 128246912 241 chr3 NM_001130081_PLD1_exon_21 171362649 171362863 214 chr9 NM_001006617_MAPKAP1_exon_8 128268538 128268746 208 chr3 NM_001130081_PLD1_exon_20 171376952 171377141 189 chr9 NM_001006617_MAPKAP1_exon_7 128305287 128305497 210 chr3 NM_001130081_PLD1_exon_19 171379799 171380010 211 chr9 NM_001006617_MAPKAP1_exon_6 128321861 128322138 277 chr3 NM_001130081_PLD1_exon_18 171392239 171392453 214 chr9 NM_001006617_MAPKAP1_exon_5 128347783 128348056 273 chr3 NM_001130081_PLD1_exon_17 171394454 171394673 219 chr9 NM_001006617_MAPKAP1_exon_4 128419879 128420128 249 chr3 NM_001130081_PLD1_exon_16 171395305 171395534 229 chr9 NM_001006617_MAPKAP1_exon_3 128432046 128432236 190 chr3 NM_001130081_PLD1_exon_15 171405110 171405424 314 chr9 NM_001006617_MAPKAP1_exon_2 128434544 128434972 428 chr3 NM_001130081_PLD1_exon_14 171406415 171406716 301 chr9 NM_001006617_MAPKAP1_exon_1 128469199 128469563 364

chr3 NM_001130081_PLD1_exon_13 171410071 171410282 211 chr10 NM_000314_PTEN_exon_1 89623144 89624355 1211 chr3 NM_001130081_PLD1_exon_12 171417484 171417666 182 chr10 NM_000314_PTEN_exon_2 89653731 89653916 185 chr3 NM_001130081_PLD1_exon_11 171426494 171426678 184 chr10 NM_000314_PTEN_exon_3 89685219 89685364 145 chr3 NM_001130081_PLD1_exon_10 171427299 171427549 250 chr10 NM_000314_PTEN_exon_4 89690752 89690896 144 chr3 NM_001130081_PLD1_exon_9 171431632 171431885 253 chr10 NM_000314_PTEN_exon_5 89692719 89693058 339 chr3 NM_001130081_PLD1_exon_8 171442435 171442628 193 chr10 NM_000314_PTEN_exon_6 89711824 89712066 242 chr3 NM_001130081_PLD1_exon_7 171443757 171443916 159 chr10 NM_000314_PTEN_exon_7 89717559 89717826 267 chr3 NM_001130081_PLD1_exon_6 171451183 171451349 166 chr10 NM_000314_PTEN_exon_8 89720600 89720925 325 chr3 NM_001130081_PLD1_exon_5 171452604 171452810 206 chr10 NM_000314_PTEN_exon_9 89724993 89731737 6744 chr3 NM_001130081_PLD1_exon_4 171453231 171453477 246 chr10 NM_019058_DDIT4_exon_1 74033626 74033868 242 chr3 NM_001130081_PLD1_exon_3 171455271 171455499 228 chr10 NM_019058_DDIT4_exon_2 74033864 74034229 365 chr3 NM_001130081_PLD1_exon_2 171455631 171455922 291 chr10 NM_019058_DDIT4_exon_3 74034402 74035844 1442 chr3 NM_001130081_PLD1_exon_1 171528138 171528334 196 chr16 NM_001199173_MLST8_exon_1 2255127 2255563 436 chr3 NM_001146156_GSK3B_exon_11 119540751 119545752 5001 chr16 NM_001199173_MLST8_exon_2 2256011 2256265 254 chr3 NM_001146156_GSK3B_exon_10 119562051 119562250 199 chr16 NM_001199173_MLST8_exon_3 2256308 2256460 152 chr3 NM_001146156_GSK3B_exon_9 119582215 119582502 287 chr16 NM_001199173_MLST8_exon_4 2256447 2256710 263 chr3 NM_001146156_GSK3B_exon_8 119595209 119595405 196 chr16 NM_001199173_MLST8_exon_5 2256985 2257161 176 chr3 NM_001146156_GSK3B_exon_7 119624551 119624749 198 chr16 NM_001199173_MLST8_exon_6 2257143 2257396 253 chr3 NM_001146156_GSK3B_exon_6 119631500 119631707 207 chr16 NM_001199173_MLST8_exon_7 2258160 2258385 225 chr3 NM_001146156_GSK3B_exon_5 119634840 119635071 231 chr16 NM_001199173_MLST8_exon_8 2258400 2258664 264 chr3 NM_001146156_GSK3B_exon_4 119642169 119642380 211 chr16 NM_001199173_MLST8_exon_9 2258709 2259468 759 chr3 NM_001146156_GSK3B_exon_3 119666064 119666248 184 chr16 NM_001039476_NPRL3_exon_12 135753 136919 1166

chr3 NM_001146156_GSK3B_exon_2 119720842 119721136 294 chr16 NM_001039476_NPRL3_exon_11 138646 138822 176 chr3 NM_001146156_GSK3B_exon_1 119812143 119813314 1171 chr16 NM_001039476_NPRL3_exon_10 138722 138938 216 chr3 NM_006218_PIK3CA_exon_1 178866260 178866441 181 chr16 NM_001039476_NPRL3_exon_9 139660 139950 290 chr3 NM_006218_PIK3CA_exon_2 178916487 178917015 528 chr16 NM_001039476_NPRL3_exon_8 142543 142773 230 chr3 NM_006218_PIK3CA_exon_3 178917427 178917737 310 chr16 NM_001039476_NPRL3_exon_7 143166 143373 207 chr3 NM_006218_PIK3CA_exon_4 178919027 178919378 351 chr16 NM_001039476_NPRL3_exon_6 148092 148349 257 chr3 NM_006218_PIK3CA_exon_5 178921281 178921627 346 chr16 NM_001039476_NPRL3_exon_5 150319 150557 238 chr3 NM_006218_PIK3CA_exon_6 178922240 178922426 186 chr16 NM_001039476_NPRL3_exon_4 160472 160654 182 chr3 NM_006218_PIK3CA_exon_7 178927332 178927538 206 chr16 NM_001039476_NPRL3_exon_3 162570 162824 254 chr3 NM_006218_PIK3CA_exon_8 178927923 178928176 253 chr16 NM_001039476_NPRL3_exon_2 180470 180640 170 chr3 NM_006218_PIK3CA_exon_9 178928168 178928403 235 chr16 NM_001039476_NPRL3_exon_1 188098 188747 649 chr3 NM_006218_PIK3CA_exon_10 178935947 178936172 225 chr17 NM_001272042_RPS6KB1_exon_1 57970356 57970736 380 chr3 NM_006218_PIK3CA_exon_11 178936933 178937115 182 chr17 NM_001272042_RPS6KB1_exon_2 57987872 57988022 150 chr3 NM_006218_PIK3CA_exon_12 178937308 178937573 265 chr17 NM_001272042_RPS6KB1_exon_3 57989994 57990215 221 chr3 NM_006218_PIK3CA_exon_13 178937686 178937890 204 chr17 NM_001272042_RPS6KB1_exon_4 58003745 58003993 248 chr3 NM_006218_PIK3CA_exon_14 178938723 178938995 272 chr17 NM_001272042_RPS6KB1_exon_5 58007427 58007585 158 chr3 NM_006218_PIK3CA_exon_15 178941818 178942025 207 chr17 NM_001272042_RPS6KB1_exon_6 58008932 58009133 201 chr3 NM_006218_PIK3CA_exon_16 178942437 178942659 222 chr17 NM_001272042_RPS6KB1_exon_7 58011480 58011671 191 chr3 NM_006218_PIK3CA_exon_17 178943699 178943878 179 chr17 NM_001272042_RPS6KB1_exon_8 58011744 58011935 191 chr3 NM_006218_PIK3CA_exon_18 178947009 178947280 271 chr17 NM_001272042_RPS6KB1_exon_9 58012503 58012711 208 chr3 NM_006218_PIK3CA_exon_19 178947741 178947959 218 chr17 NM_001272042_RPS6KB1_exon_10 58013525 58013688 163 chr3 NM_006218_PIK3CA_exon_20 178947962 178948214 252 chr17 NM_001272042_RPS6KB1_exon_11 58013774 58013952 178

chr3 NM_006218_PIK3CA_exon_21 178951831 178957931 6100 chr17 NM_001272042_RPS6KB1_exon_12 58018146 58018354 208 chr3 NM_006545_NPRL2_exon_11 50384868 50385154 286 chr17 NM_001272042_RPS6KB1_exon_13 58022716 58022929 213 chr3 NM_006545_NPRL2_exon_10 50385139 50385382 243 chr17 NM_001272042_RPS6KB1_exon_14 58023861 58027836 3975 chr3 NM_006545_NPRL2_exon_9 50385504 50385722 218 chr17 NM_001163034_RPTOR_exon_1 78518574 78519641 1067 chr3 NM_006545_NPRL2_exon_8 50385697 50385891 194 chr17 NM_001163034_RPTOR_exon_2 78599440 78599643 203 chr3 NM_006545_NPRL2_exon_7 50385907 50386044 137 chr17 NM_001163034_RPTOR_exon_3 78617477 78617660 183 chr3 NM_006545_NPRL2_exon_6 50386065 50386263 198 chr17 NM_001163034_RPTOR_exon_4 78681590 78681849 259 chr3 NM_006545_NPRL2_exon_5 50386254 50386491 237 chr17 NM_001163034_RPTOR_exon_5 78704309 78704556 247 chr3 NM_006545_NPRL2_exon_4 50386766 50386975 209 chr17 NM_001163034_RPTOR_exon_6 78727759 78728035 276

在文檔中結節硬化症：REDCap資料庫的建立以及次世代定序基因檢測 (頁 74-107)