スプライス部位の機能多型候補

スプライス部位においては，スプライシング活性測定ソフトウェアである MaxEntScan によって算出されたスコアに基づき，スプライス部位の選択に影響を与えることが予想 される多型を探索した．解析の結果，2個の機能多型候補を同定した．そのうちの1個 はすでに乾癬や他の免疫疾患との関連が報告されているものであったが（表8），1個 は本論⽂によって新規に同定された乾癬関連機能多型であった（表9）．また，その新 規機能多型候補は，その遺伝型に依存して⾃⾝の遺伝⼦発現が変化する多型（eQTL）

であった（図10）

表8：スプライス部位における既知の機能多型

Chr. Position Variant Alleles (P/R)

RAF Gene ss InterPro

annotation

Marker (P-value)

r²

6 31380000 rs199503730¹⁰⁷ G/- 0.0447 MICA 5´ ss - rs2395029 (2.00E-26⁵⁵)

0.8860

P/R, protective/risk; RAF, risk allele frequency; ss, splilce site; r², 乾癬感受性SNPと機能多型間の r-squared value

表9：スプライス部位における新規の機能多型候補

Chr. Position Variant Alleles (P/R)

RAF Gene ss InterPro

annotation

Marker (P-value)

r²

5 96245518 rs2549797 G/A 0.5199 ERAP2 5´ ss - rs2910686

(2.00E-08⁶¹)

0.8051

P/R, protective/risk; RAF, risk allele frequency; ss, splilce site; r², 乾癬感受性SNPと機能多型間の r-squared value

図10：スプライス部位における機能多型とeQTL normalized effect size 縦軸は解析した細胞・組織，横軸は機能多型とそれらを有する遺伝⼦を⽰す．セル内 の数値は各多型のマイナーアリルが遺伝⼦発現に与える影響のスコア（normalized effect size）であり，マイナーアリルの本数と遺伝⼦発現量が正の相関を⽰す場合は

⾚⾊，負の相関を⽰す場合は⻘⾊で⽰している．灰⾊で⽰す“n.s.”は，統計学的に有 意な相関がみられなかったことを⽰す．

3.3.1. rs2549797（ERAP2）

本論⽂によって新たに同定した乾癬関連機能多型候補であるrs2549797は，ERAP2遺伝

⼦第15エクソン 3´末端（第 15イントロン 5´末端）のスプライス部位近傍に位置して

いた（図11a）．この多型はスプライス部位近傍のMaxEntScan scoreを–1.440（リスク

アリルrs2549797-A）から5.463（⾮リスクアリルrs2549797-G）に変化させるものであ

った（図11b）．この機能多型候補は，乾癬感受性SNP rs2910686と強い連鎖不平衡の

関係にあった（r² = 0.8051）（図11a）．リスクアリル（rs2549797-A）からは適切なスプ ライス部位（Splice site 1）が選択されるため，遺伝⼦の全⻑が翻訳され機能的なERAP2 タンパク質が⽣成されるが，⾮リスクアリル（rs2549797-G）由来の転写産物は第15エ クソン3´末端（第15イントロン5´末端）において異なるスプライス部位（Splice site 2）

が選択されることで，エクソンが 3´末端⽅向に 45 塩基伸⻑し未成熟な終⽌コドン

（Premature Termination Codon: PTC）を含むため，ナンセンス変異依存mRNA分解機構

（Nonsense-Mediated Decay: NMD）¹⁰⁸によって分解されることが予想された．更に，

rs2549797は第10エクソン3´末端（第10イントロン5´末端）のスプライス部位に位置

する多型rs2248374と完全に連鎖していた（r² = 1.0000）（図11a）．Rs2248374-Gはス プライス部位の活性を変化させ，未成熟終⽌コドンを含む転写産物を⽣成しNMDを引 き起こすことがすでに報告されている¹⁰⁹．GTEx eQTLデータを参照すると，解析を⾏

ったすべての細胞・組織において，乾癬発症の⾮リスクアリル（rs2549797-G，

rs2248374-G）を含む遺伝型ではERAP2遺伝⼦の発現量は顕著に低下していた（図10；補⾜図2）．

図11：ERAP2遺伝⼦座における機能多型候補rs2549797

a. 5番染⾊体の乾癬感受性SNP rs2910686と機能多型候補rs2549797，ナンセンス変異 依存mRNA分解を引き起こすことが報告されているrs2248374の座位，およびヨー ロッパ⼈集団におけるr-squared value．⻘縦線は乾癬感受性SNP rs2910686，⾚縦線 は機能多型候補rs2549797，rs2248374の座位を⽰す．

b. ERAP2遺伝⼦第15エクソン3´末端（第15イントロン5´末端）の拡⼤図．⿊破線箱 はスプライス部位（Splice site 1，2）を⽰す．それぞれのスプライス部位において MaxEntScan scoreを⽰しているが，Splice site 2は機能多型rs2549797を含むため，リ スクアリル（rs2549797-A）および⾮リスクアリル（rs2549797-G）それぞれについて スコアを⽰している．

Window Position Scale chr5:

Human Feb. 2009 (GRCh37/hg19) chr5:96,206,001-96,260,000 (54,000 bp)

20 kb hg19

96,210,000 96,215,000 96,220,000 96,225,000 96,230,000 96,235,000 96,240,000 96,245,000 96,250,000 96,255,000 ERAP2

ERAP2 ERAP2 ERAP2 ERAP2 Layered H3K4Me3150

-0 _ Layered H3K4Me150 -0 _ Layered H3K27Ac100 -0 _ 100 Vert. Cons

1 -0 _

rs2910686 rs2549797

r² = 0.8051 rs2248374

r² = 1.0000 r² = 0.8051

a

b

Scale chr5:

--->

Gaps Human Chimp Gorilla Orangutan Gibbon Rhesus Crab-eating_macaque Baboon Green_monkey Marmoset Squirrel_monkey Bushbaby Chinese_tree_shrew Squirrel Lesser_Egyptian_jerboa Prairie_vole Chinese_hamster Golden_hamster Mouse Rat Naked_mole-rat Guinea_pig Chinchilla Brush-tailed_rat Rabbit PikaPig Alpaca Bactrian_camel Dolphin Killer_whale Tibetan_antelope SheepCow Domestic_goat Horse White_rhinoceros DogCat Ferret_

Panda Pacific_walrus Weddell_seal Black_flying-fox Megabat David’s_myotis_(bat) Microbat Big_brown_bat Hedgehog Shrew Star-nosed_mole Elephant Cape_elephant_shrew Manatee Cape_golden_mole Tenrec Aardvark Armadillo Opossum Tasmanian_devil Wallaby Platypus

20 bases hg19

96,245,460 96,245,465 96,245,470 96,245,475 96,245,480 96,245,485 96,245,490 96,245,495 96,245,500 96,245,505 96,245,510 96,245,515 96,245,520 96,245,525 96,245,530 C A G T G G A A A A T T A A A G T A G A T G T A G A C T T C T G T C C T A C C C T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A

S G K L N

ERAP2

S G K L N

ERAP2

S G K L N

ERAP2 ERAP2

rs2549797

2 2 1 3 1 1 1 2 1 + 6 10

C A G T G G A A A A T T A A A G T A G A T G T A G A C T T C T G T C C T A C C C T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A T G T A G A C T T C T G T C C T A C C C T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T-A A A G T C T T T G A TTA A C A G T G G A A A A T T A A A G T A G A TAT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TAT A G A C T T C T G T C C T A C C C T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TAT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TAT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T A CTC T T TAT T C T T T T C T C T T T G A T G T A A A A G T C T T T G A T C A A C A GCG G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T A CTC T T T G T T C T T T T C T C T T T G A T G TGA A A G T C T T T G A TTA A

C A G T G G A A AGTCA A A G T A G AG CT ACATT T C T G T C C TG T TC T T T G T T C TCT TT GC T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A AGT T A A A G T A G A TC CA G A C TCC TTT C C T A CTC T T T G T T C T TCT CCC T T T G A T G T A A A A G T C T T T G A T C A A - - - AG GCA A A T T A A A G T A G A TA CA GTC T T CGG T C C TG TC C TCT G T T C T T T T CCC T T TCA T G T A AGA G T C T T TAA T C A A

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = C AA AG G A ATA T T A A A G T A GG A CT A G ATT T C T G TAC T AT TC T T T G T T C T T T T CCC T TG -ACG T A A A A GCC T T T G A T C AG C A G T GAA A A A T T A A A G T A G AA CT AAA C T TGT G T C C T A CTC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C A A C A G TAG A A A A T T A A A G T AAA TCT A G A C T T C T G T CTT A CTC T T T G T T C T T T TT GC T T T G A T G T A A A A G T C T T T G A T C A A C A GC AG A A A A T T A A A G T A G A TCT A GCC T T C T G T CTT A CTC T T T G T T C T T T T CAC T T T G A T G T A A A A GGC T T T G A T C A A C A G TAG A A A A T T A A A G T A GGTCTGG A C TCC T G T- - - -CTC T T TTT T C T T TCCAC T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G TGG A TCT A G A C T T C T G T C C T AT TC T T T G T T C T T T T CAC T T T G A T G TT CACG T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T GCC CC G T TC T T T G T T C T T T T CAC T T T G A T G TTAT C - -C T T TAA T C A A C A G T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T AT TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T A GGTCT A G A C T T C TCT C C T AT TC T T T G T T C T T T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T A GGTCT A G A C T T C TCT C C T AT TC T T T G T T C T T T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T A G A TCTGG A C T T C T G T C C T AT TC T T T GCT CCT T T CAC T T T GTT G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T A G A TCTGG A CCT C T G T C C T AT TC T T T GCT CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T AAA TCTGG A C T T C T G- - - T TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T AAA TCTGG A C T T C T G- - - T TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T AAA TCTGG A C T T C T G- - - T TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T AAA TCTGG A C T T C T G- - - T TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT T GG T G G A A A A T T A A A G T AAAC CTTG A C T T C T G T C C T AT TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT CGG T G G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T AT TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C AT C A G T G G A A A A T T A A A G T AAA TCT A G A CAT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A ACG T AAA TCT A G AT AT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T GC T GA A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T AAAC CT A G A CAT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T GCA A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T AAA TCT A G A CAT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T GCA A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T AAA TCT A G A CAT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T GCA A A A G T C T T T G A T CGA C A G T G G A A A A T T A A A G T AAA TCT A G A CAT C T G T C C TG T TC T T T G T T CCT T T CAC T T T G A T GCA A A A G T C T T T G A T C A A C A GCG G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T AT TC T TC AT T CCT T T CAC T T TAA T G T A A A A G T-T T T G A T C AT C A GCG G A A A A T T A A A G T A G A TCT A G A C T T C T G T C C T AT TC T TC AT T CCT T T CAC T T TAA T G T A A A A G T-T T T G A T C AC C A G T G GGA A A T T A A A G T A G A- - - -T C T G T C C T AT TC T T T G T TT CT TCCAC T T- - - -A A A A G TT AT T G A TT T C C A G T G GGA A A T T A A A G T A G A TCT A G A C T T C T G T C C T AT TC T T T G T TT CT TCCAC T T TAA T G T A A A A G T C T T T G A T C AT C A G T G GGA A A T T A A A G T ACA TCT A G A C T T C T G T C C T AT TC T T T G T TT CT TCCAC T T TAA T G T A A A A G T C T T T G A T C AT T GG T G G A A A AATTA A G T A GGTCT A G ATT T C T G T C CCAT TC T T T G T T CCT T T CAC T T T G A T G T A A A A G T C TCT G A T C A A C A GCG GGA A A TGA A A G TC CAC CT A G A C-T CAG T C C TC T TC T T T G T T CCT T T CAC T TC CA T GC C CATG T C T T TAAC T CA C A G T G G A A A A TCA A A G T A G A TA CAAA C T T C T G TTC T ATC C T T T G T T CCT T TA AC T T T G A T G TTA A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G A TTT A G A C T T C TTT CTT AT TC T TC AT T CCT T T CAC T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T ATA TCT A G A C T T C TTTTC TT T TC T T TAT T CATGT CA TT T T G A T G T A A A A G T C T T T G A TTAC C A G T G G A A A ACT A A A G T A G A TCTTG A C T T C TTT CTT AT TC T T T G T T CCT T T CAC T T T G A T G T A A A A GCCAT T G A T C A A C A G TAG A A A A T T A A AAT A G A TCT A G A C T T C TTT C C T AT TC T T T G T T CCT T T CG - -T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A AATCG A TCTGGGC T T CC C CC C T AT GC T T T G T T C T TGT CAC T T T GGT G TGA A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G AC CT A G A C T T C TTT C C TT T TC TCT G T T CCT T TT GC T T T G A T G T A A A A G T C T T T G A T C A A C A G T G G A A A A T T A A A G T A G AC CT A G A C T T C TT C AC T AT TC T T T GCT CCT T T CAC T T T GT CG T AGA A G T C T T T GGT C A A C AAT G G A A ATT TT - - - CA GTC TG TTAT CAT AG A AT- - - -TAAC CT A A ATG T CC CTCA TT T T TAA A TGGA ATT TT - - - -AC CC T TTTAT CAT AA T AT- - - -TAA- - - - GA A G T CCT TAA T C A A

-Stop

Splice site 1 Splice site 2

Splice Site Sequence MaxEntScan score

1 AAAGTAGAT 0.25

2 (rs2549797-A; Risk) GATGTAAAA -1.440 2 (rs2549797-G; Protective) GATGTAAGA 5.463