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Human Molecular Genetics Advance Access originally published online on April 12, 2006
Human Molecular Genetics 2006 15(10):1722-1734; doi:10.1093/hmg/ddl096
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© The Author 2006. Published by Oxford University Press. All rights reserved.
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A haplotype spanning two genes, ELN and LIMK1, decreases their transcripts and confers susceptibility to intracranial aneurysms

Hiroyuki Akagawa1, Atsushi Tajima1, Yoshiko Sakamoto1, Boris Krischek1, Taku Yoneyama1, Hidetoshi Kasuya2, Hideaki Onda2, Tomokatsu Hori2, Motoo Kubota3, Toshio Machida3, Naokatsu Saeki3, Akira Hata4, Kazunari Hashiguchi5, Eizou Kimura5, Chul-Jin Kim6, Tae-Ki Yang6, Jong-Young Lee7, Kuchan Kimm7 and Ituro Inoue1,8,*

1Division of Genetic Diagnosis, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2Department of Neurosurgery, Neurological Institute, Tokyo Women's Medical University, Tokyo, Japan, 3Department of Neurosurgery and 4Department of Public Health, School of Medicine, Chiba University, Chiba, Japan, 5Kousei General Hospital, Tokyo, Japan, 6Department of Neurosurgery, Chonbuk National University, Chonju, Korea, 7Center for Genome Science, National Institute of Health, Seoul, Korea and 8Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Japan

* To whom correspondence should be adressed at: Division of Genetic Diagnosis, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan. Tel: +81 354495325; Fax: +81 354495764; Email: ituro{at}ims.u-tokyo.ac.jp

Received February 20, 2006; Accepted March 31, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The rupture of an intracranial aneurysm (IA) results in subarachnoid hemorrhage, a catastrophic neurological condition with high morbidity and mortality. Following-up on our previous genome-wide linkage study in Japanese population, we extensively analyzed a 4.6 Mb linkage region around D7S2472 on 7q11 by genotyping 168 single nucleotide polymorphisms (SNPs). SNP association and window scan haplotype-based association studies revealed a susceptibility locus for IA on a single LD block covering the 3'-untranslated region (3'-UTR) of ELN and the entire region of LIMK1. An association study with 404 IA patients and 458 non-IA controls revealed that the ELN 3'-UTR G(+659)C SNP has the strongest association to IA (P=0.000002) and constitutes a tag-SNP for an at-risk haplotype, which contains two functional SNPs, the ELN 3'-UTR (+502) A insertion and the LIMK1 promoter C(-187)T SNP. These allelic and haplotype-based associations were confirmed in a Korean population. Ex vivo and in vitro analyses demonstrate that the functional impact of both SNPs is the decrease of transcript levels, either through accelerated ELN mRNA degradation or through decreased LIMK1 promoter activity. Elastin and LIMK1 protein are involved in the same actin depolymerization signaling pathway; therefore, these lines of evidence suggest a combined effect of the SNPs in the at-risk haplotype possibly by weakening the vascular wall and promoting the development of IA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Subarachnoid hemorrhage (SAH) is most often the result of a ruptured intracranial aneurysm (IA). Patients who survive the acute phase are often left with a substantial disability and impaired quality of life as a result of major neurological deficits. The annual incidence of SAH due to ruptured IA is 18 to 23 per 100 000 (1Go–3Go), whereas the prevalence of unruptured IA detected by MR angiography, CT angiography or digital subtraction angiography in the Japanese population has been reported to be much higher, around 6–7% (4Go,5Go). The prevalence of unruptured IAs detected at autopsy ranges from 0.2–8.9% (6Go,7Go). Despite continuing improvement of diagnostic methods, treatment techniques and medical care, the 30-day mortality of aneurysmal SAH is unchanged and remains a major public health concern (8Go).

Several factors such as smoking, ethnicity, female gender (especially in menopause) and hypertension are involved in the pathogenesis of IA. Epidemiological studies demonstrate a strong genetic influence: the prevalence of unruptured IA is significantly higher (10.5–13.5%) in a Japanese subgroup with a family history of IA (4Go,5Go). The risk of SAH is four times higher in first degree relatives and six times higher in siblings of patients with SAH than in the general population (9Go,10Go). However, the etiology and pathogenesis of IA formation remain largely unknown. Several studies have attempted to identify genes contributing to the susceptibility to IA, focussing on candidates for allelic association in genes that encode mostly matrix proteins such as endoglin, lysyl oxidase and matrix metalloproteases. But they did not yield consistent results (11Go–17Go). Genetic linkage study of IA families represents a more systematic approach. In the first genome-wide linkage study of IA with 104 Japanese affected sib pairs, we identified a significant linkage to chromosome 7q11 (MLS=3.22 near D7S2472) (18Go). Although this result was confirmed in Utah families of European origin (19Go), another Japanese study failed to find this linkage (20Go). The elastin gene (ELN), a positional and functional candidate gene located within the linkage region, has been investigated for allelic association with IA, but the results were divergent (18Go,21Go,22Go). Another candidate gene, collagen {alpha}2(I) (COL1A2), has been identified as a genetic risk factor in IA patients with a family history (14Go), but there has been no subsequent replication study.

In this study, we performed systematic single nucleotide polymorphism (SNP) fine-mapping of the 4.6 Mb linkage region surrounding the peak linkage marker D7S2472. One hundred and sixty-six SNPs of 26 positional genes were initially detected and genotyped. Disease-related linkage disequilibrium (LD) was assessed using the sliding window method. Positive association with IA was found in a distinct LD block in which the 3' end of ELN and the LIM domain kinase 1 gene (LIMK1) were matched. Further analysis of this LD block revealed that the SNPs in the 3'-untranslated region (3'-UTR) of ELN and the promoter region of LIMK1 were significantly associated with IA. The functional impact and relationships of these variants were further investigated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Initial screening of the 4.6 Mb linkage region on 7q11
We initially screened 188 SNPs in the 4.6 Mb linkage region surrounding the peak linkage marker, D7S2472, in 96 familial IA patients and 160 non-IA controls of Japanese ethnicity. The SNPs were selected according to the criteria (allelic frequency >0.01, gene-based) of the database of Assays-on-DemandTM SNP Genotyping Products (Applied Biosystems, Tokyo, Japan). The 96 IA patients consisted of 78 probands from nuclear families that had participated in our genome-wide linkage study (18Go) and 18 patients with a family history of IA. The 160 selected controls, all over 50 years of age, did not harbor an IA as verified by radiological examination. One hundred and sixty-six out of the genotyped 188 SNPs were polymorphic in our Japanese population (allelic frequency >0.01) and within expectations of the Hardy–Weinberg equilibrium. In the pairwise LD analysis, mainly gene-specific LD blocks were observed (Fig. 1A). The 166 SNPs were tested for allelic association evaluated by a permutation test with 10 000 iterations. Fifteen SNPs showed significant association (permutation P<0.05), and nine were clustered within a region of ~400 kb centromeric to D7S2472 (Fig. 1B). The locus contains ELN, LIMK1 and the cytoplasmic linker 2 gene (CYLN2). To increase the statistical power to detect genetic association, systematic haplotype analyses in the 4.6 Mb region were performed using the sliding window method with two to four adjacent SNPs as shown in Figure 1C and D. Haplotypes comprising two to four adjacent SNPs with significant association (permutation P<0.05) were also clustered within the locus containing ELN, LIMK1 and CYLN2, thus confirming the single-SNP associations. After setting the threshold level for significance of the permutation P-value to below 0.01, associations were observed only in one distinct LD block, which included the 3' part of ELN and LIMK1 in both single and global haplotype-based comparisons (Fig. 1C and D).


Figure 0961
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  		Figure 1. Results for the pairwise LD analysis and the sliding window scan of the 4.6 Mb linkage region around D7S2472 on 7q11. (A) Genes, in which at least three SNPs were genotyped, are depicted in the upper panel. The pairwise LD block of the tested region evaluated by the |D'| or r2 statistics is shown. (B) Single SNP association analyses using 96 familial cases and 160 non-IA controls. SNPs are shown in single columns along a horizontal line without taking the intervals into account. The statistical value is represented by the logarithm of P-value (–log P) in the vertical line. (C and D) Haplotypes were constructed using the sliding window method with two to four adjacent SNPs. Frequencies of all estimated haplotypes were compared between 96 familial cases and 160 non-IA controls and haplotype-based association was tested using a single haplotype comparison (C) or global test (D). All P-values were generated after 10 000 iterated permutations.

 
Search for susceptibility SNPs
In the initial screening, the boundaries of the ELN–LIMK1 LD block were observed between intron 21 T(+17)C and intron 31 A(+242)G SNPs of ELN (|D'|=0.16), and between intron 2 G(–1626)C and intron 12 T(–156)C SNPs of LIMK1 (|D'|=0.57). Within the LD block, five SNPs were over-represented in IA patients with statistical significance, but all of them were intronic or intergenic. To identify the causal SNPs in the LD block, the genomic DNA of the 96 familial IA patients initially screened were re-sequenced at exons 22–33 and 3'-UTR of ELN. We also re-sequenced a 1.5 kb promoter region and exons 1–12 of LIMK1 to identify novel SNPs. Several intronic or intergenic SNPs available from the public database were also genotyped. Within the region, we confirmed a total of 16 SNPs including the six SNPs used for the initial screening. They formed highly structured LD blocks spanning from at least intron 29 of ELN to intron 10 of LIMK1 (|D'|>0.85). Although this region did not contain SNPs in the coding regions, we identified two 3'-UTR SNPs in ELN and three promoter SNPs in LIMK1. We then genotyped these five SNPs in 404 cases and 458 non-IA control individuals including the initially screened subjects. Allelic association was tested by chi-square and permutation tests with 1 000 000 iterations. All five SNPs showed significant association with IA (permutation P<0.05) as summarized in Table 1. The most significant association was observed with the ELN 3'-UTR G(+659)C SNP ({chi}2=22.5, df=1, P=0.000002, permutation P=0.000001, odds ratio=3.11). In chicken specimen, the 3'-UTR of ELN is known to play a key role in post-transcriptional regulation by binding cytosolic protein to the specific sequences (23Go,24Go). Thus, the 3'-UTR SNPs might directly affect the stability of the transcripts, despite the fact that ELN 3'-UTR sequence involving the ELN 3'-UTR G(+659)C SNP and the ELN 3'-UTR (+502) A insertion SNP (described later) is not conserved in chicken sequences. The promoter SNPs of LIMK1 might affect transcriptional activity.


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  		Table 1. Allelic association between ELN–LIMK1 SNPs and IA in Japanese population
 
Genotype-specific differences in ex vivo expression of ELN and LIMK1
To examine the functional impacts of the ELN 3'-UTR and the LIMK1 promoter SNPs on their transcripts with respect to stability and synthesis, we quantified the transcript amounts in cultured human cells of known genotypes. As cerebral arteries were not available, umbilical artery smooth muscle cells (UASMCs) from 44 umbilical cords obtained during delivery were separated and cultured. Simultaneously, isolated DNA was genotyped to obtain genotype-defined UASMC specimens (Fig. 2). UASMCs from three individuals were heterozygous for the ELN 3'-UTR (+502) A insertion SNP. Additionally, one of them carried the ELN 3'-UTR G(+659)C SNP in a heterozygous state. As for the three promoter SNPs of LIMK1, three specimens were heterozygous for all three sites. Total RNA was extracted from UASMCs of each genotype, after which ELN and LIMK1 transcripts were measured either by classical RT–PCR or quantitative real-time RT–PCR (Fig. 2). Analysis of the ELN transcripts showed that cells with the ELN 3'-UTR (+502) A insertion SNP have a reduced level of ELN transcription (Fig. 2A and B). The cells heterozygous for the ELN 3'-UTR G(+659)C SNP did not exhibit any additional impact (Fig. 2A). Although there was only one carrier of the ELN 3'-UTR G(+659)C SNP, these results suggest that the ELN 3'-UTR (+502) A insertion SNP is responsible for the reduction in the amount of ELN transcripts. Analysis of the LIMK1 transcripts showed that cells heterozygous for all three promoter SNPs of LIMK1 also have significantly reduced LIMK1 transcription, suggesting the impact of one or more of the SNPs on the promoter activity of LIMK1 (Fig. 2C).


Figure 0962
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  		Figure 2. Genotype-specific differences in ex vivo expression of ELN and LIMK1. RNA derived from UASMCs, with the genotypes shown in the figure, was subjected to RT–PCR or real-time PCR using SYBR Green to estimate the amount of genotype-specific transcripts of ELN and LIMK1. (A and B) The amount of ELN transcripts due to genotype was evaluated either by RT–PCR or by real-time RT–PCR. The ELN 3'-UTR (+502) A insertion allele is shown as A-IN, whereas the major allele at the site is shown as ‘–’. (C) The amount of LIMK1 transcripts according to the genotype was evaluated with real-time RT–PCR. Genotypes at the three sites of LIMK1 as seen in the tested cells were all heterozygous. The Welch's t-test was applied for statistical evaluation.

 
In vitro functional analyses of ELN 3'-UTR SNPs
To confirm the ex vivo findings on the effect on ELN transcripts, we first cloned three types of ELN 3'-UTR sequences into the downstream region of an SV40 promoter-luciferase gene transcriptional unit, and the effect of the insertions on the stability of the mRNA were investigated (Fig. 3A, upper panel). The constructs corresponded to three common ELN 3'-UTR haplotypes that covered 99% of the tested population (M/M, m/M and m/m—representing minor (m) and major (M) allele at the +502 and +659 sites) (Fig. 3A). They were transfected in parallel into HEK293 cells. All inserted sequences resulted in reduced luciferase activity compared with the control vector without insertion, indicating that the inserted ELN 3'-UTR sequences influenced mRNA stability. The constructs containing the ELN 3'-UTR (+502) A insertion SNP (m at +502), regardless of the +659 genotype, showed significantly reduced luciferase activity: the m/M haplotype had 48.0% and the m/m haplotype 53.5% activity compared with the M/M haplotype. There was no significant difference between the m/M and the m/m haplotypes, suggesting that the ELN 3'-UTR G(+659)C SNP does not affect the stability of the mRNA (Fig. 3B, left). A stretch of AU-rich sequence between the first and second poly(A) signals of ELN 3'-UTR was found, which might accelerate the degradation of mRNA (25Go). To eliminate the effect of the AU-rich sequence, we generated two additional reporter constructs (Fig. 3A, the two poly(A) signals of ELN were removed and a poly(A) signal derived from a pGL3 vector was used). These constructs were transfected into both HEK293 and HCT116 cells. When compared with the previous constructs, elevated luciferase activity was observed in the HEK293 cells (61.3% of the control vector) (Fig. 3B, middle). The construct containing the ELN 3'-UTR (+502) A insertion SNP showed 38.4% activity compared with the +502 major type (P=0.002, Welch's t-test). Almost identical results were observed in the HCT116 cells (Fig. 3B right). These findings strongly indicate that the ELN 3'-UTR (+502) A insertion SNP is involved in the reduction of ELN transcripts.


Figure 0963
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  		Figure 3. ELN 3'-UTR allele-dependent transcriptional regulation in HEK293 and HCT116 cells and EMSA with riboprobes containing the ELN 3'-UTR (+502) A insertion SNP. (A) ELN 3'-UTR with or without the poly(A) signal and AU-rich sequence was subcloned into the downstream region of the reporter gene pGL3-promoter vector, as described in ‘Materials and Methods’. M/M, m/M and m/m denote the minor (m) and major (M) allele at the +502 and +659 sites. +502 Major and +502 A-IN denote the major and minor allele at the +502 site. (B) Each reporter vector was transfected into HEK293 or HCT116 cells, and the firefly luciferase activity was normalized with the Renilla luciferase activity of co-transfected phRL-TK. The data are expressed as relative activity, mean+SE of six dishes. (C) DIG-labeled riboprobes containing the (+502) A insertion or the major type (A-IN and Major, respectively) were incubated with HEK293 cytoplasmic extract. The arrows point to specific binding of the A-IN riboprobe.

 
If the amount of ELN transcripts is regulated by mRNA–protein interaction as shown in chicken ELN (23Go,24Go), the ELN 3'-UTR (+502) A insertion SNP might alter the affinity for an RNA-binding protein. Two different 53 nt digoxigenin (DIG)-labeled RNA transcripts (ELN 3'-UTR sequence from +477 to +529) containing the ELN 3'-UTR (+502) A insertion or the major type allele, respectively, were synthesized to perform an electrophoretic mobility shift assay (EMSA) with cytoplasmic extract from HEK293 cells. Allele-specific RNA–protein interaction was observed in the probe containing the ELN 3'-UTR (+502) A insertion SNP after incubation with the extract (Fig. 3C). This allele-specific RNA-binding protein was fractionated with anion exchange chromatography in which the protein was eluted at about 0.4 M NaCl (data not shown). Thus, the reduction in ELN transcripts associated with the ELN 3'-UTR (+502) A insertion SNP may well be due to the binding of an unidentified cytoplasmic factor that accelerates degradation of the transcript.

In vitro functional analyses of the LIMK1 promoter SNPs
In the promoter region of LIMK1, two common haplotypes were observed, the triple minor –961A/–428A/–187T (A/A/T) haplotype and the triple major –961G/–428G/–187C (G/G/C) haplotype, both of which covered >95% of the tested population. To confirm the ex vivo findings derived from the RT–PCR study of the UASMC, we cloned these two promoter haplotypes into the upstream region of the luciferase gene (Fig. 4A, top). The effects of the promoter sequences on transcriptional activity were examined in HEK293 cells and HCT116 cells. Both inserted sequences showed strong promoter activity (Fig. 4B). The construct containing the A/A/T haplotype showed reduced transcriptional activity compared with the G/G/C haplotype (31.9% reduction, P=0.00003, Welch's t-test). To identify the critical SNP responsible for the difference in transcriptional activity, we generated deletion constructs removing the –961 site ({Delta}-A/T and {Delta}-G/C) and subsequently the –428 site ({Delta}{Delta}-T and {Delta}{Delta}-C) (Fig. 4A). The effects on transcriptional activity were examined in the HEK293 cells. Each deletion construct showed elevated luciferase activity when compared with both the three-SNP (A/A/T and G/G/C) constructs. Furthermore, the differences in transcriptional activity between alleles still remained (24.6% reduction, P=0.0006, Welch's t-test) even after the –961 and –428 sites were deleted. Similar results were also observed in the HCT116 cells (P=0.004, Welch's t-test) (Fig. 4B, right). These results indicate that the LIMK1 promoter C(–187)T SNP is responsible for the difference in transcriptional activity.


Figure 0964
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  		Figure 4. LIMK1 promoter allele-dependent transcription activity in the HEK293 and HCT116 cells. (A) The LIMK1 promoter region containing the G(–961)A, G(–428)A and C(–187)T SNPs or the corresponding haplotype (G/G/C or A/A/T) was subcloned into the reporter vector. Deletion constructs without the –961 site or both the –961 and –428 sites were generated, as described in ‘Materials and Methods’, and are shown as {Delta}-G/C or {Delta}-A/T and {Delta}{Delta}-C or {Delta}{Delta}-T. (B) Each reporter vector was transfected into the HEK293 or HCT116 cells, and the firefly luciferase activity was normalized with the Renilla luciferase activity of co-transfected phRL-TK.

 
To examine nuclear factor binding, two DIG-labeled double-stranded oligonucleotides containing the –187C and –187T alleles (Fig. 5A) were designed for EMSA using HEK293 nuclear extract. –187C allele-specific DNA-protein binding was observed after incubation with the HEK293 nuclear extract, and there was no specific competition with the –187T allele (Fig. 5B and C). EMSA was also performed for two other SNP sites, the –961G or –961A and the –428G or –428A SNPs, but there was no allelic difference in DNA–protein binding (data not shown). The TFSEARCH program showed an 8 bp sequence containing –187C within the AP2-binding motif that was collapsed after –187T substitution (Fig. 5A). AP2-enriched nuclear extract (Promega, Tokyo, Japan) showed strong binding to the –187C probe, and a supershift was observed after adding anti-AP2 antibody, but the band in the standard nuclear extract of HEK293 cells was not supershifted in the presence of the antibody (Fig. 5D). We tentatively conclude that the difference in transcriptional activity is due to different affinity of the alleles to a nuclear factor, possibly AP2.


Figure 0965
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  		Figure 5. EMSA with oligonucleotide containing the LIMK1 promoter SNPs. (A) The double-stranded oligonucleotide probe containing either the –187C or the –187T allele is shown. The computer program TFSEARCH based on TRANSFAC databases, available at website (http://www.cbrc.jp/research/db/TFSEARCH.html), was used to predict potential binding sites of transcription factors in the regulatory region. The 8 bp sequence containing –187C appears to be the AP2-alpha-binding motif. (B) HEK293 cell nuclear extract was incubated with –187C and –187T probes. The arrow points to specific binding. (C) The specific interaction with –187C probe was competed with various amounts of non-labeled –187C or –187T competitors (5-, 10- and 20-fold from left to right). (D) The –187C probe was reacted with AP2-enriched extract (AP2 ext., Promega) and the specific binding supershifted with the anti-AP2 antibody was observed. The specific binding with nuclear extract from HEK293 cells was not supershifted with the antibody.

 
Analyses of the combinational effect of the ELN and LIMK1 SNPs
We identified two functional SNPs from the ELN–LIMK1 LD block, the ELN 3'-UTR (+502) A insertion and the LIMK1 promoter C(–187)T SNPs, both significantly associated with IA. However, the strongest association was observed with the ELN 3'-UTR G(+659)C SNP having the highest odds ratio (Table 1), which had no detectable functional impact on the ELN transcript. To examine the relationships among these three SNPs, they were combined to construct haplotypes. The haplotype-based associations were tested with a 1 000 000 iterated permutation test. Four major haplotypes (each frequency >5%) were observed in 404 IA patients (Table 2). One haplotype, the triple minor (A/C/T) haplotype, was over-represented in IA patients, showing a highly significant difference in frequency between the IA and non-IA control group ({chi}2=19.2, df=1, P=0.000012, permutation P=0.000004) (Table 2). A global haplotype comparison also showed significant differences between the IA and non-IA control group (permutation P=0.00033), further indicating the involvement of the ELN–LIMK1 haplotypes in susceptibility to IA. Thus, the A/C/T haplotype both represents individuals statistically at-risk with susceptibility to IA and implicates two SNPs that reduce ELN and LIMK1 transcripts in the pathology of the disease. The most significantly associated SNP, the ELN 3'-UTR G(+659)C SNP, can be regarded as a tag-SNP for the at-risk haplotype.


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  		Table 2. Haplotype analysis at ELN–LIMK1 LD block in Japanese population
 
The association study with Korean samples
Korean samples comprising 195 IA patients and 250 controls were used to confirm the associations observed in the Japanese population. Although the two functional ELN 3'-UTR (+502) A insertion and LIMK1 promoter C(–187)T SNPs were not significantly associated with IA, the association with the ELN 3'-UTR G(+659)C SNP reached a statistical significance ({chi}2=4.89, df=1, P=0.027, permutation P=0.018, odds ratio=1.81) (Table 3). The haplotype-based association study also showed that the A/C/T haplotype was significantly associated with IA ({chi}2=5.56, df=1, P=0.018, permutation P=0.019) (Table 4).


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  		Table 3. Allelic association between ELN–LIMK1 SNPs and IA in Korean population
 

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  		Table 4. Haplotype analysis at ELN–LIMK1 LD block in Korean population
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous linkage and association studies of IA from two different ethnic groups, Japanese and Utah white populations, showed evidence of linkage in a region on chromosome 7q11, although inconsistent results were also reported (18Go–20Go). In the current study, the previously found 4.6 Mb linkage region with an LOD score >1 was extensively examined using systematic haplotype-based sliding window scan analyses and LD mapping (18Go). We were able to pinpoint a susceptibility locus to IA on a single LD block spanning the 3'-UTR of ELN and the entire region of LIMK1 in the Japanese samples. Further examination of the SNPs in the LD block using all of the available subjects with IA showed that two functional SNPs were significantly associated with IA. One of them, the ELN 3'-UTR (+502) A insertion SNP ({chi}2=4.75, df=1, P=0.029, permutation P=0.029, odds ratio=1.38), decreased the amount of ELN transcripts by altering the mRNA–protein interaction (Fig. 3). The other, the LIMK1 promoter C(–187)T SNP ({chi}2=17.2, df=1, P=0.000033, permutation P=0.000019, odds ratio=1.97), reduced the promoter activity of LIMK1 by altering AP2 or an unidentified nuclear factor binding (Figs 4 and 5). These functional impacts on the transcripts of the two SNPs were similar in both ex vivo and in vitro findings. In addition, we identified the ELN 3'-UTR G(+659)C SNP, which had the strongest association and highest odds ratio ({chi}2=22.5, df=1, P=0.000002, permutation P=0.000001, odds ratio=3.11), as a tag-SNP for the three-SNP haplotype harboring the ELN 3'-UTR (+502) A insertion and LIMK1 promoter C(–187)T SNPs (Table 2). Similar associations were observed in Korean IA samples (Tables 3 and 4). Thus, the polymorphisms in the haplotype contribute to reduction of both the ELN and LIMK1 transcripts. Although each SNP itself represents an increased risk of development of IA, the two SNPs together may exert a synergistic effect on susceptibility in considering that the strength of the vascular wall is crucial in the development of an aneurysm.

Elastin, a major component of the vessel wall, is synthesized by vascular smooth muscle cells (VSMCs), secreted as a soluble monomer, tropoelastin, and then undergoes self-assembly and polymerization mediated by cross-linking enzymes such as lysyl oxidase. Polymerized elastin transforms to insoluble polymers with a random coil structure that forms concentric rings of elastic lamellae. As cerebral arteries lack external elastic lamina, the internal elastic lamina together with the smooth muscle layer constitute the major structural support (26Go). Thus, it might be expected that the ELN 3'-UTR (+502) A insertion SNP reduces the strength of the vascular wall by decreasing the amount of elastin and leads to aneurysmal malformation. Indeed, animal models for IA have been established by treatment with elastase (27Go,28Go) or ß-aminopropionitrile, which disturbs elastin composition through degradation or by inhibiting cross-linking reactions of elastin molecules (29Go). Recently, a new biological function of elastin as a signaling molecule was noted in the pathogenesis of vascular abnormalities such as neointima formation and stenosis (30Go,31Go). Secreted elastin interacts with the surface receptor of VSMCs, activating a G-protein-coupled signaling pathway that stimulates Rho-induced actin stress fiber organization (28Go). Reduced elastin has been shown to induce subendothelial proliferation of SMCs and contribute to obstructive arterial disease in human and mice hemizygous for elastin (32Go). These findings indicate that elastin is not merely structural, but also functions as a regulatory molecule during vascular development, controlling the proliferation of smooth muscle cells and stabilizing the vascular structure. LIM domain kinase 1 (LIMK1) encoded by LIMK1 is known to be one of the major actin cytoskeletal regulators. LIMK1 requires phosphorylation by Rho kinase for its activation, indirectly regulating actin stress fiber organization by phosphorylating cofilin, rendering it inactive and unable to bind actin and mediate actin depolymerization (33Go). Thus, a synergistic effect of the ELN 3'-UTR (+502) A insertion and LIMK1 promoter C(–187C)T SNPs on susceptibility to the development of IAs may result from combined structural and regulatory quantitative impediments in the elastin signaling cascade. It is well established that ELN haploinsufficiency results in supravalvular aortic stenosis either by nonsense mutation or by intergenic or chromosomal deletion. This includes Williams syndrome, a neurodevelopmental disorder resulting from ~1.5 Mb chromosomal deletion. However, IA formation has not been reported in these diseases. It is especially interesting that in Williams syndrome both ELN and LIMK1 are deficient due to the chromosomal deletion. In a hemizygote mouse model (Eln+/–), VSMCs dedifferentiate to a proliferative non-contractile state and then migrate to the subendothelial space and contribute to neointima formation and thickening of the artery. However, it was found that Eln+/– vessels showed lower distensibility at high pressure (>100 mmHg) when compared with vessels of Eln+/+ mice, whereas the opposite was the case at lower pressure (34Go). Thus, the vascular phenotype is sensitive to blood pressure even in the hemizygous mice. For these reasons, the subtle decreases in the amount of ELN and LIMK1 transcripts together with the subsequent elastin signaling observed in the current study might readily lead to IA formation in response to microenviromental factors such as hemodynamics and atherosclerosis.

In conclusion, we have identified the ELN 3'-UTR G(+659)C SNP as a genetic susceptibility allele for IA in Japanese and Korean populations. It occurs in an at-risk haplotype containing two functional SNPs that reduce the transcript levels of ELN and LIMK1, respectively. Biofunctional analyses show that elastin and LIMK1 protein affect the stability and synthesis of vascular walls by sharing the same elastin signaling pathway. Although the effects observed throughout these experiments were significant and consistent, further replication in independent family-based and case–control samples in different populations is required to confirm the role of the haplotype in genetic risk for IA. In addition, as the carrier frequency of the ELN/LIMK1 haplotype is relatively low, it may only play a partial role in genetic susceptibility to IA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
The Ethics Committees of Tokyo Women's Medical University, Chiba University, Chonbuk National University and University of Tokyo approved the study protocols, and all participants gave written informed consent. The Japanese DNA samples for the present study were from 404 IA patients (age: 53.9±10.2 years; 254 females/150 males) and 458 controls (age: 61.4±13.0 years; 283 females/495 males). The IA patients included both familial IA patients [78 probands from nuclear families that had been used in our linkage study (18Go) and 107 patients who had a family history of IA] as well as 219 sporadic IA patients under the age of 60 at diagnosis. The presence of IA was confirmed by digital subtraction angiography, three-dimensional CT angiography, MR angiography or surgical findings. The 458 unrelated controls were outpatients of the Department of Neurosurgery of Tokyo Women's Medical University, Department of Neurosurgery of Chiba University and their nearby affiliated hospitals with diseases other than IA. The controls did not harbor an IA as verified by radiological examination and none had a family history of SAH. All were over 50 years of age to exclude most early-onset diseases.

To confirm allelic and haplotype-based associations observed in Japanese population, we performed a Korean case–control study with DNA samples of patients recruited at Chonbuk National University. The DNA samples were from 195 Korean IA patients (age: 55.2±11.2 years; 141 females/54 males) and 250 Korean controls (age: 65.7±6.4 years; 117 females/133 males). The IA patients were mainly sporadic patients except three familial cases, confirmed by surgical findings. The controls were mainly outpatients without a medical history of SAH, including a few trauma cases. All were over 55 years of age.

Selection and genotyping of SNPs
For initial screening, 188 SNPs located around the marker D7S2472 were chosen from a series of available Assays-on-Demand SNP Genotyping Products (Applied Biosystems). Genomic DNA was examined by TaqMan assay according to the supplier's manual using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems).

For the second screening, additional SNPs were picked out from the NCBI dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and IMS-JST JSNP (http://snp.ims.u-tokyo.ac.jp/) databases or identified by direct sequencing. Genotyping was then performed by direct sequencing using BigDye terminator cycle sequencing on an ABI PRISM 3700 DNA analyzer (Applied Biosystems).

Primary culture of UASMCs
We obtained 44 umbilical cords during delivery at the Department of Obstetrics of Tokyo Women's Medical University and Kosei General Hospital. All of the participants gave written informed consent, and the study was performed under the approval of the Ethics Committee of Tokyo Women's Medical University and Kosei General Hospital. Umbilical arteries were excised from the cords and cut into small pieces. UASMCs were separated using Hanks buffer containing 2 mg/ml collagenase and cultured in HuMedia-SG medium (Kurabo, Osaka, Japan) supplemented with epithelial growth factor (0.5 ng/ml), basic fibroblast growth factor (2 ng/ml), insulin (5 µg/ml), antibiotics and 5% fetal bovine serum (FBS).

RT–PCR and quantitative real-time PCR analysis
We extracted total RNA from UASMCs using TRIzol reagent according to manufacturer's instruction (Invitrogen, Tokyo, Japan). RT–PCR was performed with the SuperScript one-step RT–PCR system (Invitrogen). Real-time PCR was carried out on an ABI PRISM 7700 sequence detection system using SYBR Green PCR Master Mix (Applied Biosystems) according to supplier's manual. Copy numbers of ELN and LIMK1 transcripts were calculated by referring to standard curves and normalized by the total RNA using GAPDH as an internal control.

Transfection and reporter assays
The entire 3'-UTR of ELN containing each observed haplotype was produced by PCR synthesis. Genomic DNA of corresponding genotype was used as a template. These ELN 3'-UTR inserts were subcloned into the XbaI/BamHI-digested pGL3-promoter vector (Promega). Deletion constructs lacking the poly(A) signal sequences were also produced by PCR synthesis using the former constructs as templates. The deletion constructs were subcloned into the XbaI-digested pGL3-promoter vector and verified by direct sequencing.

LIMK1 promoter constructs containing three observed SNPs and 5'-UTR were produced by PCR synthesis using genomic DNA as templates, and ligated into the KpnI/BglII-digested pGL3-basic vector (Promega). Deletion constructs were also produced by PCR synthesis using the former constructs as templates and ligated into the KpnI/BglII-digested pGL3-basic vector.

HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and antibiotics and HCT116 cells in McCoy's 5A medium supplemented with 10% FBS and antibiotics. The cells (105 cells/well) were transfected with 0.2 µg of the each expression vector and 6 ng of phRL-TK vector (Promega) as an internal control for transfection efficiency, using Fugene-6 (Roche Diagnostics, Tokyo, Japan). At 48 h post-transfection, the cells were solubilized and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).

Statistical analyses
Differences in allelic frequencies were examined using a case–control design and the chi-square test. Haplotype frequencies for multiple loci were estimated using the expectation–maximization method (SNPAlyze v3.2 software, DYNACOM, Mobara, Japan). Additionally, the permutation test was performed to test deviation of allelic frequencies of SNPs and haplotypes (35Go). Distribution of the test statistic was estimated by evaluating the statistics for a random sampling of 10 000 to 1 000 000 iterated permutations by fixing the total numbers of both cases and controls to avoid false-positive results of multiple testing, which is incorporated in SNPAlyze v3.2 software. We calculated LD between pairs of SNPs using the standard definition of D' (36Go) and r2 (37Go).

For all ex vivo and in vitro experiments, mean and standard errors were calculated and statistical analyses were carried out with Welch's t-test or analysis of variance (ANOVA). For the luciferase assay that compared the three ELN 3'-UTR constructs (M/M, m/M and m/m, as shown in the legend of Fig. 4), post hoc comparisons following ANOVA were carried out by Scheffe's test.

Electrophoretic mobility shift assay
EMSA was performed with a DIG Gel Shift Kit (Roche Diagnostics). The nuclear and cytoplasmic extracts were prepared from HEK293 cells using a NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce Biotechnology Inc., Rockford, IL, USA). HEK293 cytoplasmic extract was fractionated by Q-Sepharose ion exchange chromatography using HiTrap column (GE, Tokyo, Japan) in 5 mM HEPES buffer, pH 7.9, containing 0.1 mM EDTA with 0–0.6 M NaCl gradient on an ÄKTA explorer (GE). An AP2-enriched nuclear extract was purchased from Promega.

The DIG-labeled 53 nt RNA transcript containing the ELN 3'-UTR (+502) A insertion or the major type alleles were synthesized using a DIG RNA Labeling Kit (T7/SP6) (Roche Diagnostics). In vitro transcription was performed using T7 promoter sequence-attached PCR products synthesized from the former ELN reporter constructs. The RNA transcript was heated for 5 min at 85°C and then cooled down to room temperature for 15 min before the following reaction. The HEK 293 cytoplasmic extracts and the fractionated proteins were incubated at 20°C for 30 min with the RNA transcripts in 5 mM HEPES buffer, pH 7.9, containing 0.5 mM MgCl2, 7.5 mM KCl, 0.5 mM DTT, 0.12 mM EDTA, in a total volume of 15 µl. Protein–RNA complexes were separated by electrophoresis on a 6% polyacrylamide gel in 0.33xTBE buffer at 4°C. RNA–protein complexes were electroblotted onto nylon membrane and the band shift was visualized according to user's manual for DIG Gel Shift Kit.

For the analyses of the LIMK1 promoter SNPs, we generated six DIG-labeled double-stranded oligonucleotides for either –961G or –961A (21 nt), –428G or –428A (21 nt), and –187C or –187T (26 nt). DIG-labeled probes were incubated with HEK293 nuclear extracts for 20 min at 25°C and separated by electrophoresis on a 6% non-denaturing polyacrylamide gel with 0.5xTBE running buffer. The DNA–protein complexes were electroblotted onto nylon membrane and the band shift was visualized according to user's manual for DIG Gel Shift Kit. For the competition assay, we pre-incubated the HEK293 extracts with unlabeled oligonucleotide probes before adding labeled oligonucleotide probes.


   ACKNOWLEDGEMENTS
 
We thank the DNA donors and the supporting medical staff for making this study possible. We are grateful to K. Eguchi, K. Ohtaka and R. Mitsumori for their technical assistance and M. Kakihara for the careful editing of the manuscript. This work was supported by Grant-in-Aid for Scientific Research on Medical Genome Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Funding to pay the Open Access publication charges for this article was provided by CREST, Japan.

Conflict of Interest statement. None declared.


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