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Research paper
Association between genetic variation on chromosome 9p21 and aneurysmal subarachnoid haemorrhage
  1. Sandra Olsson1,2,
  2. Ludvig Z Csajbok3,
  3. Katarina Jood1,
  4. Karin Nylén1,
  5. Bengt Nellgård4,
  6. Christina Jern1,2
  1. 1Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation, the Sahlgrenska Academy at University of Gothenburg, Sweden
  2. 2Department of Clinical Genetics, Sahlgrenska University Hospital, Göteborg, Sweden
  3. 3Department of Anaesthesiology and Intensive Care, Sahlgrenska University Hospital, Göteborg, Sweden
  4. 4Department of Anaesthesia and Intensive Care, University Hospital Malmö, Sweden
  1. Correspondence to Professor C Jern, Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation, SU/Sahlgrenska, SE-413 45 Göteborg, Sweden; christina.jern{at}neuro.gu.se

Abstract

Background and aim Genetic factors play a role in susceptibility to subarachnoid haemorrhage, but little is known about which genes are involved. Recently, genome wide association studies have identified the 9p21 region as a risk locus for intracranial aneurysms (IA). The aim of the present study was to examine the possible association between 9p21 and ruptured IA—that is, aneurysmal subarachnoid haemorrhage (aSAH)—in a Swedish population. There is one study showing an association between 9p21 and arterial stiffness, and arterial stiffness plays a role in the development of hypertension. Therefore, a second aim was to investigate whether a putative association is independent of hypertension.

Methods The study comprised 183 patients presenting with aSAH to the Neurointensive Care Unit at Sahlgrenska University Hospital and 366 healthy, age and sex matched population based controls. As the causative functional variant in the region has not yet been identified, a 44 kbp region on 9p21 was tagged using HapMap. Six single nucleotide polymorphism (SNPs) were genotyped.

Results Two SNPs, rs10757278 and rs1333045, showed significant associations with aSAH in univariate analyses. After adjustment for hypertension as well as for smoking, the association between aSAH and rs10757278 remained significant with an OR for aSAH of 1.42 (95% CI 1.08 to 1.87; p=0.01) for the uncommon G allele.

Conclusions These data confirm earlier results showing that 9p21 is a susceptibility locus for IA, and that this association is present in a Swedish sample restricted to ruptured IA. For the first time, it has been demonstrated that this association is independent of hypertension.

https://doi.org/10.1136/jnnp.2009.187427

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    Subarachnoid haemorrhage (SAH) is a serious condition that has a young age onset and poor outcome. The incident rate in Sweden is 12.4/100 000 person years.1 SAH is caused by rupture of an intracranial aneurysm (IA) in 85% of cases.2 The most important risk factors are smoking, hypertension and excessive alcohol intake.3 SAH occurs more often in women.4 In addition, genetic factors have been shown to play an important role in SAH. First degree relatives of SAH patients have 3–5 times higher risk of SAH than the general population.5 Furthermore, it is estimated that about 9% of first degree relatives to patients suffering from SAH/IA harbour an unruptured IA,6 7 whereas the population frequency is approximately 2%.8

    As there is heritability in IA, whole genome linkage studies have been conducted and linkage has been found to several loci. In the largest whole genome linkage study comprising 333 families, possible linkage to 4q and 12p was detected.9 There is also one genome wide association study (GWAS) on IA, on two European populations and one Japanese population, in which associations were found for 2q33, 8q11 and 9p21.10 Association between 9p21 and IA was also shown by Helgadottir et al in a candidate gene study including three different European populations.11 Interestingly, 9p21 also shows association with other vascular phenotypes, such as coronary artery disease (CAD),12–16 ischaemic stroke (IS),17 aortic aneurysms,11 and arterial stiffness.18 The molecular mechanism underlying these associations has not yet been identified. However, the finding that 9p21 is associated with aneurysms and arterial stiffness suggests that genetic variations at this locus may influence vessel properties, thereby leading to susceptibility to various vascular diseases.

    The aim of the present study was to investigate whether there is an association between genetic variants on 9p21 and ruptured IA—that is, aneurysmal subarachnoid haemorrhage (aSAH)—in a Swedish population and, if so, to evaluate whether this association is independent of hypertension as well as smoking.

    Methods

    Study population

    Consecutive Caucasian patients presenting with SAH admitted to the Neurointensive Care Unit, Sahlgrenska University Hospital, between 2000 and 2004, were recruited. During this period, of 253 patients considered, 183 fulfilled the diagnostic criteria and were willing to participate in the study. A detailed description of the inclusion criteria has been published previously.19 20 SAH was defined as symptoms suggestive of SAH combined with subarachnoid blood on CT. Only patients with aSAH, defined as an aneurysm visualised on intra-arterial angiography, were included. Patients were treated with neurosurgical clipping or by endovascular coiling. For each case two healthy Caucasian controls, matched for age (±2 years), sex and geographic residence area, were randomly selected from a population based health survey21 or from the Swedish Population Register. The latter source was used to recruit controls younger than 30 years of age. All participants gave informed consent, and when patients were unable to communicate, his or her next of kin gave informed consent. The medical ethics committee at the University of Gothenburg approved the study.

    Clinical characteristics

    Smoking history was coded as current versus never or former smoker (smoking cessation at least 1 year before inclusion in the study) and hypertension was defined by pharmacological treatment for hypertension prior to the event.

    Genotyping

    Because the causative functional variant(s) in the region 9p21 has not yet been identified, a region of 44 kbp, between position 22071397 and 22115503, on 9p21 was tagged using HapMap CEU data (Rel 23a) and HaploView 4.1 (r2=0.8 and minor allele frequency (MAF) 0.1), resulting in six tag single-nucleotide polymorphism (SNPs), rs10965227, rs1547705, rs7857345, rs1333045, rs1333040 and rs10757278. The analysed region includes SNPs that in previous studies have been shown to associate with IA (figure 1).10 11 The SNP rs1537378, recently found to be associated with the ischaemic stroke subtype large vessel disease (LVD), was also included.22 Genotyping was performed by TaqMan Assays. Primers and probes were obtained from Applied Biosystems (C_31288976_10, C_1754687_20, C_29146326_10, C_8766826_10, (C_8766795_10), C_11841860_10 and C_83169_10). Amplifications were carried out on a 384 Well GeneAmp PCR System 9700 (Applied Biosystems) and fluorescence was analysed on an ABI PRISM 7900HT Sequence Detector System (Applied Biosystems). Genotyping was performed blinded to case control status. The assay for rs1333040 (C_8766795_10) failed and could not be replaced. The five SNPs that were successfully genotyped tag 90% of the genotyped SNPs in HapMap, in the region of interest.

    Figure 1
    Figure 1

    Graphic representation of the linkage disequilibrium structure in the region 9p21 between position 22071397 and 22115503 (NCBI build 36), downloaded from the HapMap database (http://www.hapmap.org) for the CEU population. This plot shows single-nucleotide polymorphism (SNPs) assayed in the present study, and SNPs previously associated with intracranial aneurysms. *SNPs analysed in the present study; ¤SNPs analysed by Bilguvar and colleagues10; #SNP analysed by Helgadottir and colleagues.11

    Statistics

    Allele frequencies were derived from genotype data, and deviations from the Hardy–Weinberg equilibrium (HWE) were tested. Associations between single SNPs and case-control status were investigated using univariate and multivariate binary logistic regression using an additive model. The multivariate model included hypertension and smoking status as covariates. The interaction between genotype and smoking/gender was also tested in a binary logistic regression. Haplotype frequencies and associations between haplotypes and case controls status were estimated using THESIAS.23 This method uses a stochastic EM algorithm for likelihood maximisation. The individual likelihood for each haplotype was used to estimate ORs for each haplotype by comparison with a reference haplotype represented by the most frequent haplotype. Data were analysed using SPSS 16.0 (SPSS Inc), and statistical analyses were performed in a two tailed fashion. A p value <0.05 was considered significant. The studied SNPs have MAFs between 0.14 and 0.50 (median 0.30) according to HapMap (CEU population), and it was estimated that the study has 80% power to detect an OR of 1.45 at the 5% level for SNPs with a MAF of 0.3 for the risk allele. No correction for multiple testing was conducted since most SNPs are in high linkage disequilibrium (LD) and a Bonferroni correction would be too conservative.

    Missing values

    Information on smoking status was missing in six cases due to intervening death, and information on hypertension was missing in one case. Genotyping success rates were between 98% and 100%.

    Results

    Clinical characteristics for this sample have been published.19 In short, mean age of the patients and controls was 55 (range 20–81) years and 74% were females. As expected, smokers were more frequent in cases compared with controls (54% vs 20%). Furthermore, 23% of patients and 19% of controls had antihypertensive treatment. Genotype frequencies are presented in table 1. In controls, all genotype frequencies were consistent with HWE.

    View this table:
    Table 1

    Genotype frequencies

    Two SNPs showed a significant association with aSAH in univariate logistic regression analysis (table 2). The uncommon allele (G) of SNP rs10757278 was associated with an increased risk of aSAH. This association remained significant after adjustment for hypertension and smoking (table 2). For comparative purposes with earlier data, ORs for both heterozygote and homozygote subjects were calculated. The univariate ORs for aSAH were 1.25 (95% CI 0.80 to 1.95) and 1.95 (1.18 to 3.22) for heterozygous subjects and for subjects homozygous for the rs10757278 G allele, respectively. The corresponding multivariate ORs were 1.38 (95% CI 0.85 to 2.24) and 2.01 (1.16 to 3.49), respectively. Approximately 24% of patients and controls were homozygous for the rs10757278 G allele, which is in line with previous studies on individuals of European descent.11 The uncommon allele (A) of rs1333045 was associated with a decreased risk of aSAH (table 2). However, this association did not remain significant after adjustment for hypertension and smoking (table 2). In the logistic regression analysis an additive model was used. Because the causative variant is unknown, the genetic effect remains to be determined. In addition, we therefore compared genotype frequencies in cases and controls using the χ2 test. For the lead SNP, rs10757278, there was also a significant difference with regard to genotype frequencies between cases and controls (table 1). Haplotype analysis, including SNP rs10965227, rs1547705, rs7857345, rs1333045, and rs10757278, did not add any further information compared with single SNP analyses (data not shown).

    View this table:
    Table 2

    Association between single-nucleotide polymorphism (SNPs) on 9p21 and aneurysmal subarachnoid haemorrhage

    Because rs10757278 was the lead SNP in the overall analysis, we focused on this variant in subsequent subanalyses. In a sex-specific subanalysis, no significant sex-specific difference was detected. Multivariate ORs of aSAH for rs10757278 were 1.49 (95% CI 1.07 to 2.07; p=0.02) and 1.27 (95% CI 0.74 to 2.16; p=0.39) for women and men, respectively.

    Smoking is considered to be the most important modifiable risk factor for aSAH,3 and evidence of an interaction between current smoking and familial aggregation of IA/SAH has been reported.24 In our sample, 371 participants (79 patients and 292 controls) were non-smokers and 172 (98 patients and 74 controls) were smokers. An association between aSAH and rs10757278 was found in non-smokers, but not in smokers (univariate OR 1.47, 95% CI 1.03 to 2.10, p=0.03 and OR 1.37, 95% CI 0.89 to 2.11, p=0.15, respectively). However, there was no significant interaction between genotype and smoking status—that is, there was no significant difference in the effect of rs10757278 between non-smokers and smokers.

    Genotype frequencies for the additional SNP, rs1537378, are presented in table 1. In controls, the genotype distribution was in HWE. No significant association with aSAH was detected (table 2). This SNP was not included in the haplotype analysis as it is located in another LD block compared with the other SNPs in this study.

    Discussion

    This case control study with participants from Sweden provides evidence for an association between genetic variations on chromosome 9p21 and ruptured IA, thus confirming earlier data on an association between genetic variants on this genetic region and IA.10 11 In addition, this association was shown to be independent of hypertension as well as smoking.

    The 9p21 candidate region investigated in this study is 44 kbp. In addition to the SNPs showing association with IA in previous studies, this region also harbours SNPs that have been shown to be associated with CAD in several GWAS.12–16 We found the rs10757278 G allele to be significantly associated with an increased risk of aSAH. The effect of the G allele was additive, with univariate ORs of 1.25 and 1.95 for heterozygous and homozygous subjects, respectively. This finding is in line with results from a recent study on three different populations from northern Europe with different vascular phenotypes, including IA and abdominal aortic aneurysms (AAA).11 With regard to the phenotype IA, patient inclusion in this study was based on the diagnosis of IA, but approximately 87% of patients had suffered from aSAH.11 The ORs for IA in all three populations combined were 1.38 (95% CI 1.18 to 1.63) and 1.72 (1.39 to 2.13) for subjects who were heterozygous and homozygous for the G allele, respectively.11 A similar effect of the G allele was observed with regard to the risk of AAA.11 Peripheral artery disease and stroke (large artery atherosclerotic or cardiogenic stroke) also showed significant associations with rs10757278 in this study but with lower ORs.11

    There is one additional study which showed an association between 9p21 and IA.10 The Caucasian populations in this study were from Finland and The Netherlands, and in the two cohorts approximately 81% of the patients had suffered from aSAH. Associations with IA were observed for three SNPs on 9p21, rs1333040, rs10116277 and rs2383207, with ORs of 1.58–1.66 for homozygous subjects. As the lead SNP in the present study, rs10757278, is in LD with rs1333040, rs10116277 and rs2383207 (r2=0.57, 0.89 and 0.86, respectively) our results are in agreement with the study conducted by Bilguvar and colleagues.10

    As there is one study showing associations between variants on 9p21 and arterial stiffness, and there are data indicating that arterial stiffness is involved in the development of hypertension,25 26 it may be important to adjust for hypertension when assessing the association between 9p21 and ruptured IA. We found the association between aSAH and the SNP rs10757278 to be independent of hypertension and also of smoking. This is in line with studies on other vascular phenotypes that have reported independent associations between 9p21 and IS,22 27 the IS subtype LVD,22 28 CAD15 and myocardial infarction.14 15 There are, however, conflicting data from two studies that observed associations between genetic variations on 9p21 and IS or myocardial infarction, that did not remain significant after adjustment for vascular risk factors.17 29 However, the latter studies had smaller sample sizes.

    Interestingly, there is evidence of an interaction between familial aggregation of IA/SAH and current smoking.24 However, we did not find any significant interaction between rs10757278 and smoking. Similar findings could be seen for CAD.16 Thus the interaction between family history and smoking is likely to be mediated by other genes. In fact, preliminary data from a GWAS on Caucasian families from the USA show a multiplicative interaction between smoking and SNPs in the phosphodiesterase 1A (PDE1A) and type IX collagen (COL9A1) genes for IA susceptibility.30

    We also investigated rs1537378, which is located outside the previously analysed LD block on 9p21, because this SNP was recently reported to show association with the IS subtype LVD.22 In contrast with LVD, we did not detect an association with aSAH.

    The 9p21 locus harbouring rs10757278 does not contain any protein coding genes but a large non-coding RNA (ncRNA) ANRIL. As for most ncRNAs, there is limited knowledge about the function of ANRIL but expression of ANRIL has been detected in a wide range of cell types and tissues, including AAAs,16 and ANRIL was suggested as the prime 9p21 candidate gene for CAD.16 Furthermore, studies indicate that genetic variations on 9p21 influence the expression of ANRIL.31–33 Potentially interesting genes adjacent to the investigated region are CDKN2A and CDKN2B. CDKN2A encodes two proteins, p14 (ARF) and p16 (INK4a). CDKN2B encodes p15 (INK4b). These are all tumour suppressor proteins involved in proliferation, ageing and senescence.34 It has also been proposed that p16 can modulate the production of inflammatory molecules such as matrix metalloproteinase 3 and monocyte chemoattractant protein 1.35 Additionally, expression of ANRIL is positively correlated with expression of p14, and possibly also with that of p16 and p15, in both physiological and pathological conditions, suggesting a coordinated transcriptional regulation of theses genes.36 Further work is needed to determine if the association between 9p21 and aSAH is mediated through ANRIL and/or any of the mentioned genes. Moreover, we cannot exclude the fact that the SNPs of interest could be situated in distant enhancer effecting genes further away.

    This study has some limitations. Although patients were recruited consecutively, there is recruitment bias due to early death as well as the fact that patients with initial signs of an unfavourable prognosis are often treated outside the neurointensive care unit. The inclusion criterion, aSAH, also introduced some bias because some patients with a poor prognosis do not undergo angiography. The control group was recruited by random sampling from the general population in the same geographic areas as the patients, which makes the possibility of selection bias in this group less likely. In the present study, we controlled for the risk factors hypertension and smoking. However, we did not control for alcohol intake because it is difficult to obtain reliable data on this variable. The main advantages of our study are the relatively homogeneous and large patient group with confirmed ruptured aneurysms, a population based control group and a well characterised sample with regard to the risk factors hypertension and smoking.

    In conclusion, this study shows a significant association between genetic variation on chromosome 9p21 and aSAH, with rs10757278 as the lead SNP. This association was independent of hypertension and smoking.

    Acknowledgments

    The authors wish to thank Ingrid Eriksson, Ingrid Eiving, Ingrid Pettersson and Catherine Ritzén for excellent technical support. The authors also thank the Genomics Core Facility, at the Sahlgrenska Academy at the University of Gothenburg.

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    Footnotes

    • Funding This study was supported by the Swedish Research Council (K2008 65X-14605-06-3), grants from the Swedish state under the ALF agreement (ALFGBG-11206), the Swedish Heart Lung Foundation (20070404), the Swedish Stroke Association, the Rune and Ulla Amlövs Foundation for Neurological Research, the John and Brit Wennerström Foundation for Neurological Research and the Yngve Land Foundation for Neurological Research. The funding sources did not influence study design, collection, analysis or interpretation of the data, or drafting or writing of the manuscript.

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the the medical ethics committee at the University of Gothenburg.

    • Provenance and peer review Not commissioned; externally peer reviewed.