Genomics and Bioinformatics Approaches in Search of Molecular Pathomechanisms

dissertations

Approaches in Search of High-throughput genomic methods Molecular Pathomechanisms generate huge amounts data Genomics and Bioinformatics Approaches in Search of Molecular Pathomechanisms... of Molecular in Search Approaches Bioinformatics and Genomics Mitja Kurki that pose challenges for analysis of Saccular Intracranial and interpretation, requiring Aneurysm, A Complex bioinformatic methods. In this thesis, Genomics and Bioinformatics Disease to study molecular mechanisms Approaches in Search of Molecular of saccular intracranial aneurysm (sIA) disease, two complementary Pathomechanisms of Saccular approaches utilizing high-throughput genomics and bioinformatics were Intracranial Aneurysm, A Complex applied. Additionally, a bioinformatic Disease method and software was developed. Candidate genes and pathways were identified, which can serve as a basis for future research aiming to novel diagnostics, preventions, or therapies of sIA disease.

Publications of the University of Eastern Finland Dissertations in Health Sciences

Publications of the University of Eastern Finland Dissertations in Health Sciences isbn 978-952-61-1310-4 P

MITJA KURKI

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2.1 THE HUMAN GENOME

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Figure 1. Illustration of the block-like structure of the human genome caused by linkage disequilibrium. A) Typical variation in 18 individuals. Six correlating variants on the left of the recombination hotspot form three different haplotypes (out of 64 possible haplotypes) as indicated by different colors. Recombination breaks the correlation, and the next six correlating variants form two different haplotypes. B) Regions depicted in A are scattered throughout the chromosomes forming LD blocks of different sizes. The correlations shown in the chromosome are fictitious (adapted from (Altshuler et al. 2008).

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Category Count Length (basepairs) 3,323,950,079 Coding genes 20,774 Non coding genes 22,493 Pseudogenes 14,145 Gene transcripts 194,846 SNPs, indels, somatic mutations 54,965,377 Structural variants 10,266,123

Table 2. Types of variations in the human genome Type Description Single nucleotide A change of one nucleotide in DNA. polymorphism (SNP) Chromosomal Insertion, deletion, translocation or reversal of a large (> 3Mb) parts abnormality of a chromosome. Copy number variation A repetition of large (1kb - 3Mb) part of genome zero (deletion) or (CNV) more times. Interspersed element A repeating sequence of DNA, a remnant from viral genome. Some repeats contain sequence encoding viral machinery proteins required to copy and move the sequence. Simple repeat A variable number repetition of a few nucleotide sequence. Insertion or deletion A change of addition or removal of one or more nucleotides. (INDEL) Structural variant (SV) 1 kb and larger in size and can include inversions and balanced translocations or large insertions or deletions

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Figure 3. Genotype imputation. A) Illustration of the gain in power and fine-mapping provided by imputation. B) Schematic illustration of the genotype imputation process modified from the following article (Marchini & Howie 2010). See text for explanation.

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2.2 THE HUMAN TRANSCRIPTOME

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Figure 4. Illustration of a gene promoter and regulation. Chromatin consists of DNA packaged into histones. This packaging must be unwound for the transcriptional machinery to have access to the transcription start site (TSS) in the core promoter. Transcription factors bind to their specific transcription factor binding sites (TFBS) near the TSS (proximal region) or further away (enhancer) and can influence the transcriptional efficiency. Adapted from (Maston et al. 2006; Lenhard et al. 2012).

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2.3 COMPLEX DISEASES

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2.4 SACCULAR INTRACRANIAL ANEURYSM (SIA) DISEASE

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Figure 5. A) Drawing of a saccular intracranial aneurysm at a typical location in the Circle of Willis at the base of the brain (modified from www.nebraskamed.org). B) Photograph of a human saccular intracranial aneurysm taken during surgery.

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+6= 9)4,-*6 ) F ( (( ( ) ( = = 76 5 # >>?8#(( ( :& 7'=5 #&"">8# 7 6- #&"";8#( = (( ( 7+ #&"" 8# 5 ( ( 5 7,#4#( #&""@I,#( #&""@8# 5 &#@ 5 !"U( 7,#4#( #&""@8#5 ( 5 &#&1 !# 1 = 5 (# 5 ( 5 #@ &#!1 5 (# * 5 ( 5 &#&1!# 1 5 7,# 4# ( # &""@8# 5 7[ @"(<=58 57,#4#( #&""@8# F 5 = 0 5 (&"UI_!00 g/wk 21% 100 to 299 g/wk UI ;U76(5 #&"" 8# / " U ( = 76(5 #&""@I #&" "8# 5 ( (A# :#:= 5 U76(5 #&"" 8# 7+5 &" &I6(5 #&""AI #&" "8 (( ( ( 5 ( # 5 (( ) ( ( 7D&"""I #&"" IH # >>?8# '$ )'%4 ) ( ))) #

-73407)* ?+-7-2; -9 +.,*)4*).+)7 3A,*)43*3?*)7 )*,3*+36 ).> 6 8)77 ) ( # = ( 0 ( I ( I ( # ) 7)48 748 #) 5 47(:876(5 #&""@8#P

Figure 6. Schematic diagram of intracranial extracerebral artery wall structure. Adapted from (Ruigrok et al. 2005) ( )= = ( ( # ) = ( )4 ( G # 7 # >;?I3 #&""A8# 6 )= # 5 = ( 5 73 # &""AI 45 # &""?I 5 # >>>8# F ( ( 73 #&""AI 5 # >>>8#

3.3,+46 -9 ,/3 6 >+63)63 ) 76 5 # >>;I6(5 # &""@8 5( 76(5 #&"" 8 (7 #&" "I9( # >>@8 (( ( ( 5 ) ) #/ ) ( (F 5 # 9 (' = (= = G ( = = (( = ( 7 - #&" "8# = + = ) # 5 7%$ %8 $ % 7?@U 8 < $ %&( = ) 7 # &"";8#?U &U%$ % ) 7$ #&""&IT #&" 8# 1 ) :#>7>@U*)!#@W A#"8 %$ % !#A 7 #>W@#>8 ) ( 7,5 # &" 8# %$ % = "#!U 76(5 #&"" 8# ) 7;#!U87* # >>?8#+ ( >"B + (( ) ( 5 7' # &"";8# 5( ( (( ( ) 7' # &"";I 6(5 V 6 5 &""?8 = 7 !A#!W!:# ! ;F >F !#! T&&8 # ( ( ( ( ( 5 = # ( 1= ) = &""?79( # &""?8# =1 ( ( 0 ( 1= 7&?>&; '$ 8 7>&" >?@ 8 % 7;? :A&A 8 = ( 7 ` @ "1;8 D 7A>@ :;: 8# '$ 7&F!!# ?F #&!WF &# >& #!8# ) ?F ( 1= ( '$ = D # ) =1 = = 2 7;?> &&&? 8 2 2 9 ( ' = G 7A;@ >A" 8 7J # &" "8# = ( ( = D 7&&?& >"@ 8 ( 7 ?&> ;: 8 D # '$ = Y?!&""" + & * # = 5 ?F #& !F !# "F&A#!& = ?F #&!WF &# >& #!# &F!!# = = = = 9 # ) ?F '$ = '$ ( 1 = ( D # ) = ( A= =5 7 "# 1 "#@8 = 5 7J # &" 8# 7AF! #&! &F&& &" &# 8 D AF! #&! ( 1= ( = = #) = &F!!# = A ( 5= 7## 5 = 8# (( ( ( = D # ))) # 2C : :# U A#AU A# U 1 ( 5 D 7J # &" 8# ( = 4%= - ( (( ( ( ) ( ( 5 = # ( )2C= 1 = ( )= (F 5 ) 72S #&" &8# ( ! (( 7J # &" "I J # &" 8 (( @F&!#& = ( = >?>! # = &""""" # ( ( ( ) 5 = !# Table 3. Replicated genetic variants/loci linked to sIA disease

Locus Gene RAF Study Effect size Reference type 4q31.23 EDNRA 85%* GWAS OR 1.22 (95%-CI (Yasuno et al. 2011) 1.14–1.31) 8q12.1 SOX17 83%* GWAS 1.28 (1.20–1.38) (Yasuno et al. 2010) 9p21.3 CDKN2A/ 58%* GWAS 1.32 (1.25–1.39) (Yasuno et al. 2010) CDKN2B 10q24.32 CNNM2 91%* GWAS 1.29 (1.19–1.40) (Yasuno et al. 2010) 13q13.1 KL, 23%* GWAS 1.20 (1.13–1.28) (Yasuno et al. 2010) STARD13 18q11.2 RBBP8 48%* GWAS 1.22 (1.15–1.28) (Yasuno et al. 2010) 1p34.3- Many. Likely LNK - (Nahed et al. 2005; Ruigrok 1p.36.13 Potential rare et al. 2008) candidate HSPG2 (Ruigrok et al. 2006)

7q11 Many. Likely LNK - (Onda et al. 2001; Farnham Potential rare et al. 2004) candidate ELN (Onda et al. 2001; Akagawa et al. 2006) 19q13.3 Many. Likely LNK - (Olson et al. 2002; Yamada et rare al. 2004; van der Voet et al. 2004; Mineharu et al. 2007) Xp22 Many. Likely LNK - (Yamada et al. 2004; Ruigrok rare et al. 2008) 16p13.3 PKD1 Rare MEND Prevalence of sIA (Vlak et al. 2011) is 6.9 higher in ADPKD patients than in general population. 4q22.1 PKD2 Rare MEND Prevalence of sIA (Vlak et al. 2011) is 6.9 higher in ADPKD patients than in general population. RAF: Risk allele frequency; LNK: linkage; MEND: Mendelian disease associated with sIA disease; ADPKD: Autosomal Dominant Polycystic Kidney Disease; * European populations in 1000 Genomes Project '

" /-73 23.-13 3A<*366+-. <*-9+7+.2 -9 /01). 6 ,+6603 = ( ( ) 7 A8# 5 # 7 5 # &""?8 # 7 # &"">8 7)8 = = = - )) = #4 #74 #&"">8 ) = = ( 1 ( # $ # ( = ( ( ( = ( ( 7$ # &" "8#+ # ) 7++$8 7= ( 8 ( 7+ #&" "8# )= 7 A8# , = ( = F ( = = #$ 7$ #&" "8 ( ( ) = = = ( ( =( ) # ) =1( = ( ( = ( ) = ( = ( ) #+ (0 # G 7 (V4 &"";8 + 4 #) )) =1( = ) ( ( = ( ) # % Table 4. Whole-genome gene expression profiling studies of human saccular intracranial aneurysm tissue. Cases Controls Genes ↑ Genes ↓ Array Ref.

6 RAs and 4 4 AVM 263 258 Agilent Human (Krischek et al. 2008) UAs feeder 1A(v2) arteries 6 RA 4 UAs 2* 0 Agilent Human (Krischek et al. 2008) 1A(v2)

3 RAs and 3 6 STAs 172 154 Illumina Human (Shi et al. 2009b) UAs from the WG6-v2 same patients as cases 3 RAs 3 UAs 0 0 Illumina Human (Shi et al. 2009b) WG6-v2

3 UAs 3 STAs 164 996 Affymetrix (Li et al. 2009) HU133 Plus 2.0

8 RAs and 6 5 MMA 8 123 Affymetrix (Pera et al. 2010) UAs GeneChip Human Gene ST 1.0

8 RAs 6 UAs 1 31 Affymetrix (Pera et al. 2010) GeneChip Human Gene ST 1.0

12 RAs 10 UAs 10 4 Affymetrix (Marchese et al. 2010) U133A

12 RAs 5 22 8 Affymetrix (Marchese et al. 2010) U133A STA/MMA

from the same patients as cases RA: Ruptured saccular intracranial aneurysm; UA: Unruptured saccular intracranial aneurysm; STA: Superficial temporal artery; AVM Arteriovenous malformation; Middle meningeal Artery; * Did not specify if the genes were upregulated or downregulated. & 2.5 CHALLENGES AND BIOINFORMATIC SOLUTIONS FOR INTERPRETATION OF RESULTS FROM HIGH THROUGHPUT ANALYSES

(1 ( G ( ( 1= ( ( #* ( 5 ( ( # ( = F ( # = ( # F = = 745 #&" "I #&" &I6 #&" I #&"":8# 1 = ( = ( ( = ( 1= 5 ( 2C # ( ( ( #) G ( ( (1 ( = ( G ( ( #(# = 2C #

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

Figure 7. Illustration of a Gene Ontology tree. Annotations can be depicted as directed acyclic graph where directed edges (arrows) go from broader term to more specific term. / (2 / ( ( ( ( ( (1 ( ( ( = - ( B ( #) 2 / ( = # = ( ( = 7 = 8#) (( ( ( = ( ( = ( 7(?8#/ = 2 2 7 228 $CJ 7 V 2 &"""I # &" &8# 22 $CJ ( ( = # 22 = ( = 7#(# 8 ( ( 7#( G 8 ( ( = 7#(# C ( ( = 8 ( 7#(# * 8 7#(# 8# = ( ( ( = 6 7* # &" 8# 6 = = 6 - 1== = # * 6 Y !""

= ( Y:!"" = = > # = ( = = #$ ( 7===# ( #(8 = :" ( ( = #

. Example of a more elaborate pathway including pathway topology. (http://www.genome.jp/kegg/) (http://www.genome.jp/kegg/) topology. pathway including pathway elaborate a more of Example . 8

Figure " ),/8); ).)7;6+6 13,/->6 / = = ( ( = ( = - #% ( ( # = = :? = 7 ( # &"">8# = = #7 #&" &8

3868382 %)%%#%&!''"!@!1&&@ = ( 7#(#( ( = # 8 ( = # ( = ( (#(# *1 F B # ( 5 ( ( ( - ( ( ( # 5 ( = ( ( = # ( ( - ( # % = ( # ( ( ( ( ( F # ) =( ( #(# = ( ( = ( ( ( = # = ( F ( = =( ( ( ( ( = # ( 1= ( ( = ( ( # +( ( ( 7 = ( = 1 "#"A> "#"@ 8 - ( ( ( # ( = 7 ( (( ( ( = 8#) ( ( = ( = ( ( ( = G # ( 5 = ( ( = 5 = ( = ( (( = # = ( 5 = = (# )) G ( ( ( #

3868383 '!% !'@!1&&@ ) 1 ( ( G 7 1 ( ( 8( 78 = #+ ( = ( =575 V &"">8# ( 1 ( 1 a1 (#(# # # ( 1 ( = = = 1 = 1( ( # ( #(# #75 V &"">8#' ( 1 ((( ( = # (( 1 ( 7 ( # &"":8 5 ( = ( 5( 1 7 #&""@8 ( ( = ( #$ ( 1 ( = ( = ( = F = (( ( ( 72G5V 1 &"">8# ( = ( ( = 1 1 ( 7#(# < 8 (( ( #2 ( ( ( G#/ = ( = ( 7 # &""@8# ' ( = ( #7% #&"">I'V &""?8#/ = 7!!"""( [! "" 82 728 5 ( 7#(# (8 ( 7C( (1 8 7 # &""@I + # &""!8# G1 7)G #&"">8# 1 # = ( ( ( # G ( ( = # G ( ( = 5 ( ( = # (1 = ( ( ( ( =5( #

3868384 ("#""1@&@#'01@!1&&@ /6 ( = ( = ( ( ( ( = 22 6 # = ( 7$8 5 ( (( = # $ 1 = ( ( ( 1 # 76 ^ #&""AI #&"">I%( #&"";8#) 7)8 $) ( 7%( # &"";I # &""># ) ( 1 7 $ $8 ( ( $ ( ( ( = #$ = 1 7 ) )8 $ ( = # ( = 1 ( ( ( ( = ( = ( = #) G 1 ( G () 7%( # &"";I #&"">8# $ ( ( ( ( 5 2 5 = #/ $ ( ( ( 1 ( ( # 5 = ( 791+ #&"">8# ' " /)773.236 ).> 7+1+,),+-.6 -9 <),/8); ).)7;6+6 $ = (( ( ( # >@U 2 / ( = # 5 ( = 76 # &""?8# = = ( 791+ # &"">8# = ( ( = # + ( ( = 7D #&" 8# = ( ( F( #=>"U ( ( ( 7C ( #&""?8# ( (( ( ( ( ( # )) ( = 5 = ( ( ( #

" ),/8); ).)7;6+6 +. 6,0>+36 % ( ( 2C ( ( = ( ( ( 1= ( ( 2C #= ( = ( ( ( ( ( # = ( 1= ( 1= 7C ( #&" "Ia ( #&" "I6 #&" &8#/ 5 ( ( = ( ( '$ ( # '$ ( ( '$ """ = ( # ( =5 2C = = 5 ( ( = 1 ( = ( # = ( = # = ( ( ( 5 ( ( = 7 ( ( '$ = ( = 1 8 #2C = = ( 1 ( = ( # % ( = 7C ( #&" "8#

" " .93**+.2 *3207),+-. -9 >+993*3.,+)77; 3A<*3663> 23.36 / ( (F ( # C ( ( 1 F ( =#* ( =( G ( ( ( ( 7 &#&# 8# ( ( 5 = = ( ( # = ( D 79 #&""?8 7C ( # >>:8 F ( ( 7C V &""A8# = = ( ( ( 7a ( # &"">8# * F ( = ( ( 7 V 9 5 &"":I 6 # &"":8 ( ( 7 V &" &8# = = 7 V &" &8# / ( 7 &#@#&8 ( ( ( = # ) ( ( = ( ( ( 7' # &"">8#

" $ 706,3*+.2 +. /+2/,/*-02/<0, 23.-1+4 >),) 1+.+.2 ) (1 ( ( ( 7#(# ( 8 ( # * ( ( ( ( 7= 8 ( #/ ( )) ( ( ( # ( ( & #( ( #(#7 #&""?8 ( #(#7 #&""?8#

" & 07,+<73 ,36,+.2 ?0*>3. 5 ( (( 1= ( = 1 ( = # 5 = ( 5 ( 1 = = 7 8 1 ( ( # F ( = ( - (= ( <&"7##1`"#"@8#)=5&" (= ( - = # ( (1 ( (# ) 9 7 1 8# == 7#(#'$ 4% ( 8 9 7## ( 8 =#) ( 7 #&""@I*V%( >>A8# + ( 5 ( ( 1= 7C68 #9 C6 # 7%68 ( ( #%6 (- #%6 9 - ( % 6 79 - V ( >>@8 =#(# =1( ( = 7#(# )8# C P

( = 078( ) 78 ( ( ( ) = ( ( = # 5 = ( ( ) ) = = = ( 1 ( ) # = ( ( = =1 ( ( 1= G ( (1 5 # /3 6<34+9+4 )+16 -9 ,/3 6,0>; 83*3

8- +>3.,+9; 6+2.)77+.2 <),/8);6 )4,+53 +. *0<,0*3> /01). 6 8)776 )6 4-1<)*3> ,- 0.*0<,0*3> -.36 06+.2 8/-73 23.-13 ,*).64*+<,-13 <*-9+7+.2 ).> *37),3> ?+-+.9-*1),+46 • C ( ( ( = ( ) = G ( ( # F 5 = ( = ( ( )= #) ( = ( ( # &8- >3537-< ) .-537 13,/-> ).> 6-9,8)*3 9-* +.,3*<*3,+.2 >+993*3.,+)77; 3A<*3663> 23.3 63,6 ,- 2)+. 90*,/3* +.6+2/, +.,- ,/3 6+2.)7+.2 <*-436636 -*4/36,*),3> ?; ,/3 >+993*3.,+)77; 3A<*3663> 23.3 63, +. ,0>; B+1 C • C G ( ( ( )# ( ( ( = =# • C ( ( = ( # • C G ( ( ( ( ( ) # • C 5 = # !C - +>3.,+9; .-537 23.3,+4 9)4,-*6 )9934,+.2 60643<,+?+7+,; ,- 6 >+63)63 ?; 23.-13 8+>3 )66-4+),+-. ).)7;6+6 ).> 9-77-80< *3<7+4),+-. +. ,/3 /+2/*+6= <-<07),+-. -9 +.7).> • C ( 5 ( ) ( ( 1= ( ( =F ( 5 # • C ( 5 ( ( # • C ( = ( D # • C ( ( ) ( # D ! I IκI I

4.1 INTRODUCTION

( ) ( ( ) #) 5 #7 5 #&""?8 # 7 # &"">8 7)8 = = = - )) = # ( ( = = )= #4 74 #&"">8 ) = = ( 1 ( # $ 7$ # &" "8 ( ( ) = = = ( = ( ) # + 7+ #&" "8 "( A = ( ( ) # )= #9 ) ) ( ) # ( )= = )= #) = ? )= = 55 = = #

),3*+)76 ).> 13,/->6

),+3.,6 ).> +6603 )1<736 ? ) = ( ( 57("PA8 73 #&""AI3 #&"":I # &"":I 45 # &""?8# = # = 1G F ( 5 ' ( ) 9 5# &> ) = = 7 @8# * '( / ( / (( ' ( 5 * # Table 5. Patients, Saccular Intracranial Aneurysm Samples, and Methods

Sample Sex Age Location Rupture Time from Microarray qRT-PCR of sIA of sIA Rupture (h) No Years

1. F 60 MCA - + -

2. F 64 ICA - + -

3. M 47 MCA - + -

4. F 37 MCA - + -

5. M 42 MCA - + +

6. F 62 MCA - + +

7. M 56 PCoA - + +

8. F 65 MCA - + +

9. F 56 MCA - - +

10. M 42 MCA - - +

11. F 59 MCA - - +

12. F 54 MCA + 16 + -

13. F 46 ACoA + 96 + -

14. M 58 MCA + 24 + +

15. F 71 ACoA + 216 + +

16. F 52 ICA + 168 + +

17. F 32 MCA + 3 + +

18. F 83 MCA + 2.6 + +

19 M 73 MCA + 3.6 + +

20. F 69 MCA + 6.7 + +

21. F 53 MCA + N.A. + +

22. M 70 MCA + 14 + +

23. F 57 PCoA + 6.4 - +

24. F 58 MCA + 12 - +

25. F 44 MCA + 11 - +

26. F 53 MCA + 24 - +

27. F 47 ACoA + 5.2 - +

28. M 41 ACoA + 9.1 - +

29. F 62 PCoA + 72 - +

F = female; M = male; MCA = middle cerebral artery; PCoA = posterior communicating artery; ACoA = anterior communicating artery; ICA = internal carotid artery; N.A. = not available.

6-7),+-. -9 1 ).> +4*-)**); ;?*+>+D),+-. 6'= = G6( 7) ( * * 8 ( B 7 # &""&8# F F 6'= G = ' %'%1 """ 7' % ( ) C ( %=8 ( 1F 6'= &:"<&?" [ #? `&#"= #6' == ? = G ( &1 #9 "" ( 6'= G 1 %'#%'= 1 6'#$ 6' 7&" (8 = G 2 !! $ &#" 2 * # = = 7 A""8 7 2 * !"""8 ( B #

+4*-)**); ),) .)7;6+6 + = = 6 = &#># 76 % * &"">8 9 &#A# 72 # &""A8# % = = 5( #&&&:= 9 * *% & ( ( 7% #&""@I (V4 &"";8#= ;;?? ( # 6 + (= G = ( ( ( 7C #&""A8#' (= 5 ( = 7@"U = 8#4 &# ?#! 5(7 &""A8 = ( = ( ( ( ( 9 ( # 9 ( ( ( = ++ = 7 + 6 5( 7, V6 &""&88 1 F # 9 - (79 - V( >>@8 = - ( - $`"#"@= ( #

" 0.4,+-.)7 .)7;6+6 -9 +993*3.,+)77; A<*3663> 3.36 = ( = ( ( # ( ( 2 2 = 2/ 65( &# 79 V &""A8 G ( 7%,)%87 ( #&"">I% D #&""!8 = # ; ;?? ( = 5( ( # ( ( ( ( ( = ( ( ( 79 V &""A8# 9 - ( 79 - V ( >>@8 = - ( - $`"#"@= ( # ( ( ( = = = %,)%7 ( #&"">8= 2 % # ) ) = ( # = = 7&#:1 : 8 7&A1& : 8 ( # = ( = #) $ = - ( = 9 - ( 79 - V ( >>@8 $ ` "#"@ = ( # 5 ( +65(7, V6 &""&8= ( ( ( = ( ( = #

4.1.6 )!@!" *).64*+<,+-. )4,-* .)7;6+6

( = @""" ( = = C 2 , 7a # &""@8# @B = = # ( ( ( F = F = ' ( ( # $` "1@= ( #

4.1.7 )7+>),+-. -9 +4*-)**); ),)

@( = F ; : ) 7 @8# 9 @"" ( 6' = %' ( 7$( + C 8 +1 +4, 7+9) + 8# H 6' =1 = 1 1 ( ( 7 9 * * 8= 9)$6)+;;""F % 7 9 8 ( ( 7 91 8 &!4# ( = *%AA 7 "" @!!"Ac 8 )+$ 7 "" ; @@?c 8 ,2 7 "">"""@@c 8 '6 7 " "A&! !c 8 '6& 7 "" @!@@"c 8# ( = ( = (( ( # + = # ? 6'= ( G 6' # = C 1 $`"#"@ = ( #

4.2 RESULTS

C ? )= ( ( ( @? @: # C= ;;?? ( #) )= :?:( = ( ( ;A"= = ( = )= 7 ( 8#( ( 7*%AA )+$ ,2 '6 '6 98 = F 7(>8#@= ( '6 ( # %

Figure 9. Comparison of expression of 5 selected genes in 16 ruptured and 7 unruptured saccular intracranial aneurysm (sIA) wall samples by quantitative real-time polymerase chain reaction (RT-PCR). Gene expression ratios in the RT-PCR study and in the microarray study (in parentheses; 11 ruptured and 8 unruptured sIA wall samples) are presented. The y-axis indicates in arbitrary units the expression level of each gene normalized by ribosomal RNA expression levels. SDs are shown on top of each bar. P values are indicated below the gene names.

) )=( ( # = ( ( = ( # ( 26+ ( ( (7( "8#

Figure 10. Ten ruptured saccular intracranial aneurysm (sIA) wall samples arranged according to the expression levels of all 17 788 genes studied (see Materials and Methods). The 5 samples resected < 24 hours after the rupture are denoted by E (early), and the 5 samples resected >= 24 hours are denoted by L (late). The time elapsed in hours from rupture to the resection is shown in front of the rectangles. The axes are in arbitrary units. & ( ( ( ) = ( = = 5 1 15 7468 ( ( ( 5 ( 7 :8# ( 7 ( 8 ( ( = ( # / ( &

UPREGULATED GENES

Gene Ontology (GO) P FDR Count biological processes GO ID* value** *** OR **** Size*****

chemotaxis GO:0006935 2.60E-14 5.66E-11 7 30 125

immune response GO:0006955 5.70E-14 6.26E-11 4 49 333

response to external stimulus GO:0009605 1.00E-13 7.41E-11 3 74 651

inflammatory response GO:0006954 1.40E-13 7.52E-11 4.2 46 296

locomotory behavior GO:0007626 1.50E-11 6.46E-09 4.5 35 208

response to stress GO:0006950 3.60E-09 1.31E-06 2.1 99 1224

response to other organism GO:0051707 4.70E-08 1.49E-05 5.7 18 87

positive regulation of tumor necrosis factor 12 production GO:0032760 5.80E-08 1.60E-05 7 6 7

locomotion GO:0040011 1.30E-07 3.18E-05 4.7 20 114

cytokine production GO:0001816 2.50E-05 5.49E-03 3.8 16 108

phosphate metabolic process GO:0006796 5.30E-05 1.06E-02 1.8 66 893

positive regulation of interleukin-6 production GO:0032755 5.90E-05 1.08E-02 42 4 6

regulation of cell proliferation GO:0042127 1.80E-04 3.10E-02 1.9 44 550

intracellular lipid transport GO:0032365 2.60E-04 3.55E-02 21 4 8

neutrophil chemotaxis GO:0030593 2.70E-04 3.55E-02 12 5 14

regulated secretory pathway GO:0045055 2.70E-04 3.55E-02 12 5 14

Protein amino acid phosphorylation GO:0006468 2.70E-04 3.55E-02 1.8 47 613

regulation of cytokine biosynthetic process GO:0042035 3.90E-04 4.55E-02 4.2 10 61

Purine ribonucleoside monophosphate biosynthetic process GO:0009168 3.90E-04 4.55E-02 11 5 15

KEGG biological process KEGG ID P value FDR OR Count Size

Cytokine-cytokine receptor interaction 4060 8.50E-06 7.82E-04 2.7 31 245

toll-like receptor signaling pathway 4620 1.10E-05 7.82E-04 4 17 94

hematopoietic cell lineage 4640 1.70E-05 8.10E-04 4.3 15 78

epithelial cell signaling in Helicobacter pylori infection 5120 5.80E-05 2.03E-03 4.3 13 67

fructose and mannose metabolism 51 3.30E-04 8.81E-03 5.6 8 33

leukocyte transendothelial migration 4670 3.80E-04 8.81E-03 3 16 112

Gene Ontology cellular compartment GO ID P value FDR OR Count Size

membrane GO:0016020 2.50E-07 5.30E-05 1.5 325 6028

vacuole GO:0005773 3.30E-07 5.30E-05 3.5 26 193

Arp2/3 protein 11 complex GO:0005885 9.30E-07 9.80E-05 0 5 6

cytoplasm GO:0005737 5.10E-05 3.80E-03 1.4 329 6428

integral to plasma membrane GO:0005887 6.00E-05 3.80E-03 1.7 72 1053

NADPH oxidase complex GO:0043020 2.20E-04 1.18E-02 22 4 8

extracellular space GO:0005615 3.20E-04 1.43E-02 1.9 39 497

membrane raft GO:0045121 5.60E-04 2.23E-02 3.9 10 66

lysosome GO:0005764 8.10E-04 2.85E-02 3.1 13 108

cytosol GO:0005829 1.30E-03 4.09E-02 1.6 55 827

Proton-transporting V- type ATPase, V0 domain GO:0033179 1.50E-03 4.40E-02 22 3 6

DOWNREGULATED GENES

Gene Ontology

cellular compartment GO ID P value FDR OR Count Size

nucleus GO:0005634 1.40E-05 4.81E-03 1.5 235 4487

intracellular part GO:0044424 5.90E-05 1.04E-02 1.8 79 1487

costamere GO:0043034 9.60E-05 1.11E-02 31 4 7

adherens junction GO:0005912 5.90E-04 4.36E-02 3.6 11 82

tight junction GO:0005923 6.30E-04 4.36E-02 5.6 7 36

* identification code; ** p-value uncorrected for multiple testing; *** false discovery rate, p-value after multiple testing correction (see Materials and Methods); **** number of differentially expressed genes in each biological category; ***** total number of genes assayed in the present study in each category.

Table 7. Enriched transcription factor binding sites in the -5 kb promoter regions of the 686 upregulated genes. Transcription factor Number of TRANSFAC® HGNC Number of binding sites binding in human * ** sites genome P value*** Family

ELF1 ELF1 411 11549 1.66E-08 ETS

PEA3 ETV4 714 21887 1.66E-07 ETS

ETS2 ETS2 413 11968 3.64E-07 ETS

ETS1 ETS1 728 22501 3.87E-07 ETS

HIF1 HIF1A 304 8663 3.13E-06 -

Rel/ank NFKAPPAB65 RELA 96 2232 7.99E-06 yrin

Transcription factor name at *TRANSFAC® (www.gene-regulation.com) and ** HUGO Gene Nomenclature Committee gene symbol; *** nominal p-value.

4.3 DISCUSSION

) = ? ) = = = = # ) = = = 5 ( ( ( 7 ("P D =8# ( ( = (( 46 ( ( ( '159 ) 5 )= #/ ) = ( ( #

$ =1( ( ( ( ) = ( 7 &#A#@=8#) = :?: ( ( ;A" ( = ( ( ) = # ( ( 5 G = 1 1 ( # ) ( )= #= ) = ( ( # = AA &; = 5 7 5 # >>>8 = ( = ( # 3 73 # &""A8 A& &A )= 5 (( 5 )= # ( ( ) = ( 5 # * F = #

= = 5 ( 7* # &"">8 ) =# ) ( ( =1 1= = ( 46 ( ( 7% G # &""A8 *4 7$ # &"":8,2'%$'159 ( ()4?*T*6A$T!'6& $4% )*+ #7* #&"">8% #7% #&""A8 ;&( ( = = = ! ( = ( ( )= # ( (( - #!?1@ B #

) = 7( V &"">8 = 75 5 # &"">8# / (( ( = ) =# ) = = 5 1 5 46 ( ( 5 ( 7 :8 '159 ( ( 7 ;8 1 ( ( ( 7:8#

) ( 5 *% :! 5 *26!979&""?8= = ( ( ( #= )= #$ ( ( 7+2 5 V /B' &"";8 7( 8# *26!9 = 7 G &" &8 F ( ( 7J # &" "8 ( = 5 = 7 # &""@8# ) *26!9 7 #&""&8 (( ( )= #

Figure 11. Upregulated genes in selected cellular signaling pathways in a neutrophil granulocyte, hypothesized here to infiltrate a ruptured saccular intracranial aneurysm wall. Neutrophil signaling pathways are adapted from the following references (Reichel et al. 2006; Nizet & Johnson 2009; Rommel et al. 2007; Tanaka et al. 2003). Red boxes indicate proteins of the upregulated genes; white boxes indicate proteins of the nondifferentially expressed genes. NCF1 gene in the grey box was not included in the array. Many of the signaling pathways are also used by macrophages. NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP+.

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a # &""@8# ) ) #(# 7, 5 #&"";8#'159 5 ) 75 #&"";8 = ( '159 7+ 5 #&"";8#

4.4 CONCLUSIONS

? ) = = 5 ( ( ( = ) = # ( ( = (( 46 ( ( ( '159 ) 5 )= # F ( = ) = F 6' ( # ' E $% "% "%#!!%%%

5.1 INTRODUCTION

2 = ( ( = # = ( 7%8 ( # ( 7 8( ( % ( ( = ( 2 / ( 72/87 #&"""8# ( 7 = #8 F ( # 7/68 7 = &#@#&8# ( 5 % ( # % ( 5 ( = = F ( ( ( = ( # ( ( (( ( = ( 7$5 # &""@I % D # &""!I + # &""A8 ( ( 7+ #&""A8#* ( (( ( ( ( ( ( #

% = ) 7)8 = 4# ) 4 ( ( ( # = F5 ( 7( &8# 4 2 / ( 7 # &"""8 ( ( ( ( # ( % ( # 4 ( ( = &41( ( 79 8 ( ( ( 9 78 6% 76 #&"":8# 1( ( ( ( # ( ( ( 2/ # 4 ) 7)8= ( ( 2/ 2/ ( ( # ) ( ( ( # ( ( ( (( ( # == 4 + ( &# ) = = 4 = # = G ( &>! = 5 +$7+$8 = # = # = 0 8 ( 5 = +$ 7*698 ( +$ ( I &8 = 5 = ( ( 7+$ ( (8 = 5 = ( = = ( # =1 G ( = 7 )8= )# = 1 G (4 4 5 ( ( # (( ) = 4 ( G ( # & C 4( ( ( 0 2/1 ( B 1 2/1 ( 2/d 71 # &""A8 ( * ( %,)%7% D #&""!81 ( 2/1 ( ( 2 * 7* 1G #&"";8 2 7 # &""@8# = 4 = ( #

(%2 (!% 2

!& !!"&&"!% !!"&&"!%& & ! ! ! !

#!%

! !& ! ! !

!& ! ! ! !

%$(!&$%&!*'%&$%

!$ &$ % !#!!&)!&) !$ &$ % & &

) &! "!&$"#$"%%%& !

Figure 12. The flow diagram of TAFFEL analysis. From the top: the list of genes given by the user is annotated by GO and TF information from Ensembl (20) and cisRED (12) databases. The genes are clustered separately in parallel, based on GO and TF annotations (for simplicity only the TF clustering tree is shown). In each resulting cluster, the enrichment of both GO and TF annotations is determined, providing a basis for suggesting implications between the biological processes and their regulator molecules. 5.2 RESULTS

" 364*+<,+-. -9 ,/3 13,/-> ).> ,--7 4 1 ( 7$5 #&""@8 ( ( ( ( 2/ #( ( =( ## - =( # (( ( 2/ ( # ( = ( G # ( G G ( ( ( ( 7( !8# % ( ( # G ( =( ( ( ( ( = ( = # ( = - = ( ( 1 5 # 5 ( ( # G 5 ( ( G ( #) ( = ( 7 8# ( (( G # (2/ ( K2/ M ( K M# ( 4 2/ ( = 72/8= (# ( = G ( ( # ( ( ( ( # F 7 K4 1M =8 ( ( # = F 4 = ( 7 K* 1M =8# ( = ( G ( # ) ( ( ( ( 4 = # ) ( ( ( ( 2/ # 4= ( ( = = 2/ # &05"3I 133"(5&10 ( ( ( (B 2/ (# 9 ) 1 ( ( ( ( ( = ( ( #

Figure 13. TAFFEL user interface. The clustering trees represents the clustering result for the DE genes after 4 hours of forskolin treatment in HEK293T cells. The genes have been clustered by the GO terms (left) and TFs (right). The topmost box represents the whole gene set without clustering. Below that, each level represents clustering to two, three, or more clusters. The green outline indicates the cluster number selection by AIC and blue by dAIC. The clusters obtained from the IEA analysis with FDR p < 0.1 are highlighted with the light blue background on the right side of cluster box. The best intercorrelating clusters (cell morphogenesis cluster in the GO tree and COUP cluster in the TF tree) between the trees are connected with the bold line. Information at bottom shows enriched annotations (left list) and cluster genes (right list). Positive regulation of biosynthetic process related cluster is selected in the picture.

" 5)+7)?+7+,; ).> *0..+.2 ,/3 <*-2*)1 4 D C = ( D : = ' 9 ( 7===# #(8# + H4 7===# F#8 #6 (4F D 6 :#4(1( 42$4 0<<555##< < <#

" ;<+4)7 ).)7;6+6 97-8 == 4 = ( &# ( 4 (2/ # 2/ =( ( # = )* 2/ = G ( = ( ( # ( ( # () 1 # ( G # ( G = (( ( ( 2/ = = ( 2/ < # ( T+4#

" .)7;6+6 -9 9-*6=-7+. 39934, -. # 43776 4 ) 5 &>! +$ ( ( = 1 # *69 = = ( 5 # +$ ( ( $ )) #

" " 602236,6 .-537 /;<-,/3636 -9 6+2.)7+.2 134/).+616 )4,+53 +. ,/3 *0<,0*3> 6 8)77 A>? A> ( )=(= 4 2/ 9# > 2/ 9 ( #C = ( 7 `"#"@8 7)8 ( &05"3I 133"(5&10# ( ) ( &05"3I 133"(5&10 = 7?8# = ( ( ( #+ 5 ( ( = +1 1 ( = # +1 ( #(# G 1

( 7+8 ( 72^ # &" &8# 1 ( +& 7 8 ( +1 7 # &""&8# + G 7 ( &"":I 2 # &""?I + V ef &"";8# + G = ( (

72 #&""?8# (+1 =( (1 + = 1 G 72 #&""?I* = #&" "8# # ) ( 7 VC&" IVa (&" &I% #&" "8#/ ( BD( ( ( (( # 11 $&6TA = 7 5 #&" 8# ( (( ( F = ( - ( )= # ) ( = 1 '1 7 * ' *8 7%6 Z"#"!;8# ' * ')*5 5 * A@" ( *J$ 795V+&"" 8# &*J$1 ( G (( = ( (( ( 7 A8# ' * 795 V +&"" 8 = 1( ( ( ' * 5 745 #&" !8# ) = ( = 1( ( # = = 2/ "((P)15&10 "((P231'" 5&10P0!P 13$0&@5&10H (11!P 7"44"(P )132%1$"0"4&4 1 7%6 Z"#"! 8# 1 5 = ( ( (( 7* # >>;I4G5 # &""A8# 1 ( ,1 # ,1 1 1 - ( G #,1 21β ( (

( ( = (( 76 #&""?8# ) = ( 2/ = = ( 4 &05"3I 133"(5&10 = # 5 = +& 4!= ( 7%6 Z;#@1:8# +& = 1 ( # +& 7C ( #&""!8 5 51 = +& 7a # &" &8# = = ( G = 73 # &""A8# / (( +& ( )= #

Table 8. Statistically significant clusters in up-regulated (sIA↑) and down-regulated (sIA↓) genes in ruptured intracranial aneurysms. CLUSTER column indicates the clustered dataset, annotations used for clustering (either GO or TF) and the size of the cluster, respectively. ANNOTATION column indicates enriched GO terms and TF annotations from TRANSFAC in each cluster. P and P LIST columns indicate Benjamini-Hochberg FDR corrected Fisher’s exact test p- values for the enrichment of the annotation in the cluster and in the gene list, respectively. N and N LIST columns show the number of genes associated with the annotation in the cluster and in the gene list.

CLUSTER ANNOTATION P P LIST N N LIST

sIA ↑ GO 58 cation transport 3.8E-08 1.8E-01 17 23

ion transport 3.8E-08 3.7E-01 18 26

metal ion transport 1.2E-05 4.2E-01 12 16

…

protein amino acid 4.5E-4 9.6E-03 16 35 phosphorylation

regulation of protein kinase 1.0E-02 1.5E-01 7 12 activity

G-protein signaling, coupled 2.9E-02 1.6E-01 4 5 to IP3 second messenger (phospholipase C activating)

regulation of Ras GTPase 7E-02 1.5E-01 4 6 activity

MTF-1 4.8E-02 4.7E-01 13 30

ATF-1 4.8E-02 6.5E-01 9 17

sIA ↓ GO 22 nervous system development 1.3E-11 1.3E-02 17 32

generation of neurons 2.1E-06 2.8E-01 8 10

cell development 2.1E-06 2.8E-01 10 17

…

blood vessel morphogenesis 3.5E-04 2.8E-01 5 6

cell migration 4.3E-04 7.2E-01 4 4

Tal-1 3.1E-02 1.0E+00 4 5

AR 9.4E-02 9.8E-01 6 17

sIA ↓ GO 49 organic acid metabolic 1.3E-08 2.8E-01 13 13 process

carboxylic acid metabolic 1.3E-08 2.8E-01 13 13 process

oxidation reduction 1.2E-07 2.8E-01 14 16

…

lipid metabolic process 5.3E-07 4.4E-01 13 16

carbohydrate metabolic 2.8E-3 5.5E-01 7 9 process

amino acid metabolic process 3.2E-03 5.2E-01 5 5

NF-1 3.7E-02 9.8E-01 14 30

" $ -1<)*+6-. -9 ,- -,/3* 13,/->6 G ( ( 2'6/67$5 #&""@8%,)%7% D #&""!8 2/71 # &""A8 2/9 7+ # &""A8 2 +$$ 7%F # &""&8 2+ 7a( #&""!8/ 7%( #&""!8 27 #&""@8 = #(# = # 2 * 7* 1G # &"";8 ( 4 5 ( = = ( = ( ( 7## (8# 4 ) = = = (( ( ( ( ( ( # C ) = 7 )) 8 = ( = #) = = ( ( ( #

5.3 DISCUSSION

C 7%8( 1 1( ( = ( # ( ( = 6% = ( 76 #&"":8# = ( ( 7 5 #&"":I ' # &""@8# + ( ( <2/ ( ( # - (2/ 5 = ( ( 7 2/ 8 76 # &""?8 ( 7 8 # 2/ = =1 ( ( 7 6%8 ( 7 &""?8# ( ( 7 ( 8 ( # ( F 5 ( #) ( 1 ( 2/ # ( 1 ( =5 = ( ) # )* 5 ( = # ( ( 7 = 75 #&""&I5 #&""&8#) = ( ( = # 4 % ( 5 &>! = ( #(# +$ 2/ *69 # 1 +$ 1 $ 16 =5 ( # *69 ( ( ( $ ))# ) 1 ( 7 )8 = 4 ( # 4 (( ( ( ( A ( AP AP +& # ( ) = ( = # #

5.4 CONCLUSIONS % ) = 4 1 ( = ( ( #/ = ) 4 ( ( = # = ( #) = = ( ( ( # 5 1 &>! 4 = =15 = *69 ( ( = # = ) = ( 4 G F 5 # ( (( 4 ( = #

5.5 MATERIALS AND METHODS

" " ..-,),+-. >),) 6-0*436 ( (( 4 2 / ( 7 #&"""8 7%&""? 8 75 # &""?87 @! 8# GP "("$04# 4 ( ( 2/ # 4 9 6% 76 # &"":8 ( ( = F ( ( ( # 7 8 6'* 7+ &""!8 D$6 79 # &""?8 # ) 4 = 1 ` "#"" = F # C 7 >8 7 A8 GP"("$047 A8#

" " 3.3 4706,3*+.2 13,/-> ) ( ( = ( 72/ 8 #= ( #) G = =7( 8 7 8# & (= ' 1 ( G 7'+874V ( >>>8 # ( ( ( 7 8 7. #&""!8#9 # # ( ( 7 2/ 8 = 7$5 #&""@8#

" " 3734,+-. -9 .01?3* -9 4706,3*6 ) ( = = ( 4 55) * 7)*8755 >;A8 #)* 5 ( ( ( ( G (15 # % = ( 7 #(# 7* V+ &""@I4 #&"">I ( #&""!88# 72/ 8 ( ( 7$5 #&""@8#9 ( ( # = = = = # = )* )*== 1 ( = [& =4 # = ( )* =( )* ( # ) )* 7 8 ( = )*# = ( )* ( ( =( # @"U 2/ 5 ) 7 "46(54 8#C ( = ()* = ( = ( #

" " 706,3* 6,),+6,+46 ( 4 ( B #/ = (# (1 ( ( 9 - 1( % 6 7%68 79 - V( >>@8#

1 4 = # ) G 7% %8 7)8 = (#1 ( ) 0K P8P&4P015P!"2"0!"05P1#P5%"P$"0"P$3162P9P%1)1$"0"164P&0PP5"3)4M#% = 8 9 1 ( ( # 1 % = 0 KP 5"3)P 8P &4P 015P !"2"0!"05P 1#P $"0"P $3162P 9P %1)1$"0"164P &0P P 5"3)4M# 9 8 ( ( 1 15 ( = ( # % ( # 1 ( B = 9 - 1( # ( = 1 ( = # = = - ( ( ( = = ( ( #

" " " *-4366+.2 -9 >31-.6,*),+-. 1+4*-)**); >),) 63,6 2 72&":" 2 !! 8 ( 5 &>! = = 2 / 72/8 G ( 6+ # 5 1 &>! 7 8 = A ( # CB 1 = 9 - 1( = # % = ( = (# $1 ` "#"@ ( [ #&@ :> ( # C ( ? ) = ( ( ) 5= ( 21 !! $ &#" 7 8# = 6+ G ( CB 1 = 9 - 1( 1 # 2 = 1 ` "#"@ = ( ( # A>? A> ( )=(#

6.1 INTRODUCTION

!U 7 ) 8 ( 7,5 # &" I 6 5 # >>?8# >@U ( ) 7 )18 ( 5 ( 7 2- #&"";8# A1> """""= = 7( #&"">8 = ( D 7 6- #&"";8# ) 5= ( 5 ( 5 (7,#4#( #&""@8 "U ) 76 5 # >>;I #&" "I6(5 #&"" 8# ( = 72C8 7 D ( 1= ( 8= )0AF! #&!7/6 #&&87J #&" I4= #&" &8I?F #&!W F &# 7/6 #&?8I>& #!7/6 #! 8I "F&A#!&7/6 #&>8I &F&&7/6 # :87J #&" 8I !F !# 7/6 #&"8I ?F #&7/6 #&&87J #&" "8I >& #!7( #&""?I #&" &87 @ )))8# = :# UA#AU A# U 1 ( 5 D 7J # &" 8# ) 2C &F!!# = 0 = ( 2C79( #&""?8 ( ( =12C7J # &" "8 2C 5 = D 7J #&" 8#

( G ( # % G F 5 ( =F = F 7 """ 2 $- * &" &8# !:+ 7===# #(8I (7%87$ # >>>8#C G =F )1 # ) 2C = = """2 # ) ) ) = ( ) 76(5 # &""AI +5 &" &I #&" "8# # = G ) # C ;:" ) &@ ! = ?@? ) A"A? # = % ; ; ) !""A = F 5 G = 7( A8# ) = G &F!!# 79( #&""?I5 #&" "IJ #&" 8 ) 5 % #

Figure 14. Study design. The Finnish discovery and replication cohorts represent a population with an over two-fold increased risk of subarachnoid hemorrhage from ruptured saccular intracranial aneurysm (sIA-SAH). The Finnish discovery cohort was intentionally enriched with familial sIA patients, and 9.4M genotyped and imputed variants were studied. The loci with p < 5E-6 were replicated in an independent and unselected Finnish sIA sample. The allele frequencies and effect sizes of the replicated variants in Finland were finally compared to a continental European population using a Dutch sample. The sIA-SAH risk is not increased in the Netherlands (‘general risk’ in the figure).

$ RESULTS

$ )63 56 4-.,*-7 ).)7;6+6 +. +..+6/ ).> 0,4/ 6)1<736 ( ;:" (1 5 )7A"U8 &@ !( # !"A!>> ( 7J # &" "8 ( """ 2 $- 7! + &" & 8 = F >!@>&! # H 1F 7HH8 1 ( 7Z #"A8 7 ( ( 8# & # :@!A:A ( 1= ( 7`@ "1?8 A `@ "1: 7 ( 8# C ;'$ ( @ (7`@ "1:8 ?@? ) A"A? 78#'$ = `"#"@= ) 7 ( 8 = )>& #!7 !!!"A&I

/6 #! Z :#! "1;8 !F !# 7 ! &A:&!I /6 "#?? Z "#" 8# ( 1= ( & # 7Z"#&>8#) 1 = '$ ( 1= ( ` @ "1?7>8#= 0&F&!#!7 ;A>;&; AI/6&# >@U*) #:?1&#:!Z;#A "1 F *&#!@U8@F! #!7 !? :& :I/6 #>&*) #@!W&#A" Z #;A "1?*&#">U8 :F&A#&7 ;@" ?& !I/6 #>;*) #:1&#A!Z&#&@ "1 "* &#@!U8#/ = >& #!7 !!!"A&I/6 #! *) #& W #A&Z #? "1 *A&#!U87>8# = F G = % (; ; ) !""A 78# (( ( &F&!#! @F! #! :F&A#& = F 7 #:1!#>U8 ' 7 >8# = G = ( 0@F! #!7 !? :& :I/6 #! *)"#>?1 #;@Z"#"A@*!#?;U8 :F&A#&7 ;@" ?& !I/6 #@*)"#>?W&#!Z"#"!A * &#>&U8# >& #! % 7 !!!"A&I/6 #!&*) # ;W #A>Z!#A& "1:*A;#?:U8#) 1 % &F&!#!7 ;A>;&; AI/6 #?>Z #A& "1>8 @F! #!7 !? :& :I/6 #::Z!# ; "1?8 :F&A#&7 ;@" ?& !I #?;Z;# "1 8 = ( ) ( 1= ( 7>8#

Table 9. Five loci with genome-wide significant association to saccular intracranial aneurysm (sIA) disease in the Finnish and Dutch samples.

All meta- Case vs. control analysis Finnish discovery Finnish replication Finnish meta-analysis Dutch replication analysis

Case Ctrl Case Ctrl Case Ctrl Case Ctrl SNP* Gene OR P OR P OR P OR P OR P MAF MAF MAF MAF MAF MAF MAF MAF

rs74972714 LYPD6 (40kb)** 3.4% 1.7% 2.73 3.43E-06 4.9% 2.8% 1.88 4.11E-06 4.2% 2.4% 2.10 7.41E-11 1.7% 1.6% 1.04 4.37E-01 1.89 1.42E-09 (C/A) 2q23.3

rs113816216 FSTL4*** 4.5% 2.1% 2.31 8.26E-07 3.2% 2.1% 1.60 2.57E-03 3.8 2.1% 1.92 1.74E-08 4.5% 3.9% 1.30 4.53E-02 1.66 3.17E-08 (G/C) 5q31.1

rs75018213 EPM2A*** 5.1% 2.7% 2.11 3.44E-06 4.2% 2.4% 1.85 2.85E-05 4.6% 2.5% 1.97 2.25E-10 2.9% 2.3% 1.50 3.39E-02 1.87 7.14E-11 (A/G) 6q24.2

rs1333042 CDKN2B-AS1 50% 43.2% 1.32 3.01E-06 48.1% 41.7% 1.30 6.30E-07 49% 42.3% 1.31 1.81E-11 54.3% 47.9% 1.32 3.42E-06 1.31 6.71E-16 (G/A) † 9p21.3

rs919433 ANKRD44 48% 42.8% 1.25 2.53E-04 48.6% 44.6% 1.18 1.01E-03 48.3% 44% 1.21 2.15E-06 41.8% 33.2% 1.43 9.77E-09 1.27 2.20E-12 (A/G) ‡ 2q33.3 ***

rs12472355 ANKRD44 47.8% 42.7% 1.24 2.89E-04 48.8% 44.3% 1.21 2.23E-04 48.3% 43.7% 1.23 4.84E-07 39.1% 31% 1.39 1.05E-07 1.27 1.87E-12 (A/C) ‡ 2q33.3 (30kb)**

* For each variant major allele / minor allele and locus are given.; ** The variant’s distance (kb) to the nearest gene is given.; *** Located in the intron of the given gene.; † The previously reported 9p21.3 locus (Helgadottir et al. 2008; Yasuno et al. 2010).; ‡ The previously studied 2q33.3 locus with inconclusive evidence (see Materials and Methods).

Table 10. The locus with a genome-wide significant association to the number of saccular intracranial aneurysms (sIA) per individual in the Finnish samples. All meta- Association to sIA count Finnish discovery Finnish replication Finnish meta-analysis Dutch replication analysis

Case Ctrl Case Ctrl Case Ctrl Case Ctrl SNP* Gene RR P RR P RR P RR P RR P MAF MAF MAF MAF MAF MAF MAF MAF

rs150927513 (T/A) 7p22.1 RADIL* 6.0% 3.6% 1.95 8.86E-08 7.0% 5.2% 1.39 8.36E-4 6.5% 4.6% 1.60 4.92E-09 0.3% 0.3% 0.97 4.82E-1 1.59 6.08E-09 (4894744 bp)

* For each variant major allele / minor allele, locus and base pair position are given. ; * Located in the intron of the given gene

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! " #$% $ &' (()*(+,)$$%!$&'++-)*+.) Table 11. The Finnish and Dutch study samples used in the association analysis of saccular intracranial aneurysm (sIA) disease.

Finnish discovery Finnish replication Dutch replication

Cases Controls Cases Controls Cases Controls

N 760 2,513 858 4,048 717 3,004 Women 443 (58%) 1,454 (58%) 532 (62%) 2,182 (54%) 492 (67%) 1,135 (38%) Familial sIA 300 (40%) - 51 (6%) - 100 (15%)* - sIA-SAH 561 (74%) - 587 (68%) - 658 (92%) - Mean age (yrs) 50 52 52 40 54 68 Number of sIAs mean 1.54 (1-8) - 1.46 (1-6) - 1.26 (1-7) - > 2 242 (32%) - 257 (30%) - 127 (18%)** -

* Unknown familial sIA status for 35 patients.: ** Number of sIAs not known for 16 patients

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Figure 15. Regional association plots of the five identified saccular intracranial aneurysm (sIA) loci in the combined Finnish samples and the Dutch sample. Association p-values (–log10 scale, y-axis) of variants are shown according to their chromosomal positions (x-axis). Blue lines indicate the genetic recombination rate (cM/Mb). Figures A-C present the loci identified in the case vs. control analysis at 2q23.3, 5q31.3, and 6q24.2, respectively. Figure D presents the 7p22.1 locus associated to the sIA count per patient. Figure E presents the 2q33.1 locus with inconclusive previous evidence. Purple rectangles indicate the most significant variant in a) the Finnish discovery sample and, along the dashed line, its p-values in b) the combined Finnish samples (META FIN) and in c) all samples (META ALL). Adjacent variants in linkage disequilibrium (r2; EUR populations, 1000 Genomes March 2012) to the index variant are shown in colours indicating their r2 levels (r2 box in each figure).

6.3 DISCUSSION

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6.4 MATERIALS AND METHODS

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7.1 HIGH-THROUGHPUT METHODS IN THE STUDY OF COMPLEX DISEASES

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Oligonucleotide mRNA, Oligonucleotide baits Relatively Can only detect Microarrays miRNA, to capture mRNA cheap, mature known transcripts, profiling of printed on chips bioinformatics biological disease designed based on methods for interpretation of tissue. known transcripts in quality control results can be human reference and data- challenging. genome. analysis. RNA-seq mRNA, High throughput Higher dynamic More expensive than miRNA, sequencing of range than microarrays, less profiling of transcripts. microarrays, can mature statistical disease detect novel methods for quality tissue. transcripts, control and analysis, identification of biological sequence interpretation of variants in results can be transcripts. challenging. SNP microarrays Genetic Assayed variants Very good Detection of known studies to designed based on genotyping variants only, most identify known variations quality, arrays identify only genetic risk and LD in human relatively cheap, common variants, variants / genome (e.g. mature translating findings loci. HapMap). statistical into methodology pathophysiological

and software. understanding is often challenging.

Next generation Genetic Protein coding parts Affordable high Misses non-coding exome sequencing. studies to of genome are coverage variants, can detect identify captured using sequencing of coding variants of (causative) capture kit, which is protein coding only known genetic risk designed based on parts of transcripts. variants / known transcripts. genome, robust loci. Next generation single nucleotide sequencing of variant and captured DNA. small INDEL detection methodology, moderate IT requirements, coding variants can be easier to interpret biologically. Next generation Genetic Next generation Identification of Especially high- whole-genome studies to sequencing of full variants coverage sequencing sequencing identify genomic DNA. everywhere in is expensive, larger (causative) the genome structural variant genetic risk instead of pre- detection still variants / loci selected parts, unreliable, very high robust single data storage and nucleotide analysis variant and requirements, small INDEL interpretation of non- detection coding parts of methodology genome is very challenging 2

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calculating pathway existing pathway significance resources GWAS pathway Identification of possibly Helps to put the Permutation based analysis weak but coordinated genetic findings in analyses statistical signal among biological context, aid computationally genes belonging to a in discovery of demanding, relatively common pathway. genomic loci with recently developed and weaker risk effects. no clear consensus exist about best methodology. G

7.2 QUEST FOR MOLECULAR PATHOMECHANISMS OF SACCULAR INTRACRANIAL ANEURYSM DISEASE

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~~dissertations~~

~~| 202 | Mitja Kurki | Kurki | Mitja | 202~~

~~Mitja Kurki Genomics and Bioinformatics~~

~~Publications of the University of Eastern Finland Dissertations in Health Sciences~~

~~isbn 978-952-61-1310-4~~