Regulation of CARD8 Expression by ANRIL and Association of CARD8 Single Nucleotide Polymorphism rs2043211 (p.C10X) With Ischemic Stroke
Background and Purpose—ANRIL has long been considered as the strongest candidate gene at the 9p21 locus, robustly associated with stroke and coronary artery disease. However, the underlying molecular mechanism remains unknown. The present study works to elucidate such a mechanism.
Methods—Using expression quantitative loci analysis, we identified potential genes whose expression may be influenced by genetic variation in ANRIL. To verify the identified gene(s), knockdown and overexpression of ANRIL were evaluated in human umbilical vein endothelial cells and HepG2 cells. Ischemic stroke and coronary artery disease risk were then evaluated in the gene(s) demonstrated to be mediated by ANRIL in 3 populations of Chinese Han ancestry: 2 ischemic stroke populations consisting of the Central China cohort (903 cases and 873 controls) and the Northern China cohort (816 cases and 879 controls) and 1 coronary artery disease cohort consisting of 772 patients and 873 controls.
Results—Expression quantitative loci analysis identified CARD8 among others, with knockdown of ANRIL expression decreasing CARD8 expression and overexpression of ANRIL increasing CARD8 expression. The minor T allele of a previously identified CARD8 variant (rs2043211) was found to be significantly associated with a protective effect of ischemic stroke under the recessive model in 2 independent stroke cohorts. No significant association was found between rs2043211 and coronary artery disease.
Conclusions—CARD8 is a downstream target gene regulated by ANRIL. Single nucleotide polymorphism rs2043211 in CARD8 is significantly associated with ischemic stroke. ANRIL may increase the risk of ischemic stroke through regulation of the CARD8 pathway.
Atherosclerosis is one of the most common causes of both ischemic stroke and coronary artery diseases (CADs).1 Stroke is a leading cause of morbidity and mortality in China and other parts of the world.2 Ischemic stroke accounts for ≈87% of all strokes3 and is caused by both genetic and environmental factors. To date, large-scale genome-wide association studies have identified several risk loci for ischemic stroke, including HDAC9, PITX2, ZFHX3, 9p21, PRKCH, and NINJ2.4 However, most loci have small effects and may explain a small proportion of the heritability of ischemic stroke. Nearly 50 risk loci for CAD have been identified by genome-wide association study but explain ≈10% of the heritability of CAD only.5 Therefore, more genetic factors for ischemic stroke and CAD remain to be discovered.
Single nucleotide polymorphisms (SNPs) at the chromosome 9p21 locus were found to be associated with both ischemic stroke and CAD by genome-wide association study.4,5 The 9p21 locus contains an annotated noncoding RNA, termed ANRIL (antisense noncoding RNA in the INK4 locus). ANRIL is considered as a prime candidate gene for atherosclerosis at the 9p21 locus.6 First, SNPs associated with ischemic stroke and CAD (rs10116277, rs7865618, rs564398, rs496892, and rs7044859) within the 9p21 region are located within the ANRIL gene.7 Second, ANRIL is expressed in cell types and tissues that are involved in atherosclerosis. Third, several studies investigated the association of ANRIL with 9p21 SNP genotypes and showed a correlation of ANRIL expression with atherosclerosis severity, even though the direction of the effects is still in dispute.6,7 Moreover, the risk alleles of rs10811656 and rs10757278 disrupted a binding site for transcriptional factor STAT1, and STAT1 in turn regulated ANRIL expression.8 The STAT1 signaling pathway mediates responses to inflammation on stimulation of the proinflammatory cytokine interferon γ.9 These results supported the notion that ANRIL might play a role in the inflammatory response and atherosclerosis. The molecular mechanism by which ANRIL mediates atherosclerosis is unknown. However, as a long noncoding RNA, ANRIL may play its role in atherosclerotic processes by influencing the expression of other genes.
In this study, we identified CARD8 as a downstream gene of ANRIL and assessed the association between CARD8 SNP rs2043211 and ischemic stroke or CAD in Chinese Han populations.
Materials and Methods
Analysis of Expression Quantitative Loci for ANRIL SNPs
To identify potential downstream genes regulated by ANRIL, we analyzed ischemic stroke- and CAD-associated SNPs rs10116277, rs7865618, rs564398, rs496892, and rs7044859 in ANRIL.7 These SNPs were shown to influence the mRNA level of ANRIL.7 We performed expression quantitative loci analysis for these SNPs by searching the database at University of Michigan Center for Statistical Genetics (http://www.sph.umich.edu/csg/liang/imputation/). These studies identified several genes whose expression may be associated with SNPs evaluated in our study. We chose to evaluate CARD8 for the other identified genes because of its increased expression in atherosclerotic lesions.10
Cell Transfection and Quantitative Real-Time Polymerase Chain Reaction Analysis
Details of cell transfection and quantitative real-time polymerase chain reaction are described in the Materials in the online-only Data Supplement. The sequence of ANRIL siRNA was as follows: 5′-GGAATGAGGAGCACAGTGA-3′. Plasmid pcDNA3.1-ANRIL (NR_003529.3) was synthesized by GENEWIZ (Beijing, China). The sequences of primers used for quantitative real-time polymerase chain reaction are listed in Table I in the online-only Data Supplement.
All study participants were selected from the GeneID database.11 Diagnostic criteria for ischemic stroke, CAD, and related factors are described in detail in the Materials in the online-only Data Supplement. This study followed the principles outlined in the Declaration of Helsinki and has been approved by local institutional review boards on human subject research. Written informed consent was obtained from all participants.
Genotyping and Statistical Analysis
Details of isolation of genomic DNA, SNP genotyping, and statistical analysis are described in the Materials in the online-only Data Supplement.
ANRIL Regulates Expression of CARD8
Five 9p21 SNPs rs10116277, rs7865618, rs564398, rs496892, and rs7044859 are located within ANRIL and affect the expression level of ANRIL mRNA.7 By searching a public expression quantitative loci database (http://www.sph.umich.edu/csg/liang/imputation/), we identified 87 genes whose expression may be associated with 1 of the 5 9p21 SNPs (Table II in the online-only Data Supplement). One of the 87 genes, CARD8, became a strong candidate gene downstream of ANRIL because it also showed differential expression in a preliminary microarray analysis comparing HepG2 cells treated with ANRIL siRNA to those transfected with control siRNA (data not shown).
To verify that CARD8 is a downstream gene regulated by ANRIL, HepG2 cells were transfected with ANRIL-specific siRNA to knockdown the ANRIL expression (NC siRNA as negative control) and used for quantitative real-time polymerase chain reaction analysis. Compared with NC siRNA, ANRIL siRNA successfully reduced its own expression by ≈83% (P<2.0×10–5) and the expression of CARD8 by ≈55% (P<2.4×10–4; Figure [A]). Similarly, human umbilical vein endothelial cells transfected with ANRIL-specific siRNA showed significant reduction of ANRIL by 70% (P<1.5×10–5) and CARD8 by 48% (P<6.7×10–4) when compared with cells with NC siRNA (Figure [B]). These data suggest that ANRIL regulates the expression of CARD8. Consistent with the siRNA studies, HepG2 cells transfected with pcDNA3.1-ANRIL for 48 hours showed a 57-fold increase in ANRIL mRNA expression (P<5.84×10–5) and 1.6-fold increase in CARD8 mRNA expression (P<3.1×10–3; Figure [A]). Because of a difficulty in transfection of the specific line of human umbilical vein endothelial cells under this study with plasmid DNA, we did not obtain any data on the effect of ANRIL overexpression on CARD8 in human umbilical vein endothelial cells.
Characteristics of Study Subjects
Two independent cohorts were used to assess whether CARD8 SNP rs2043211 is associated with ischemic stroke. The discovery cohort for the ischemic stroke study consisted of 903 cases and 873 controls enrolled from Hubei Province in Central China. The replication cohort for the ischemic stroke study consisted of 816 cases and 879 controls enrolled from hospitals in Northern China (Table 1). The case–control cohort for the CAD study consisted of 772 patients with CAD and 873 controls from Hubei Province in Central China (Table 1). Patients with ischemic stroke or CAD had a higher prevalence of conventional risk factors, including smoking, history of hypertension, diabetes mellitus, and a lower level of high-density lipoprotein cholesterol (Table 1).
Statistical power analysis was performed for all 3 cohorts before each study. Each cohort had >90% of power to detect an association between rs2043211 and ischemic stroke or CAD with an odds ratio (OR) of ≥1.20 at the nominal type I error rate of >0.05 and a minor allelic frequency of >0.43 for rs2043211 in the Chinese population (HapMap HCB data).10
Significant Genotypic Association Between SNP rs2043211 and Ischemic Stroke in 2 Independent Chinese Populations
The genotyping data for rs2043211 did not deviate from the Hardy–Weinberg equilibrium in the control group (P>0.05). In the discovery population for the ischemic stroke study, no significant allelic association was detected between rs2043211 and ischemic stroke (P-obs=0.077, P-adj=0.092; Table III in the online-only Data Supplement). Similarly, no significant association between rs2043211 and ischemic stroke was detected in the replication cohort or the combined discovery/replication population (P>0.05; Table III in the online-only Data Supplement).
Genotypic association analysis was then conducted because this type of study can provide genetic insights into the association under different inheritance models (additive, dominant, or recessive). Interestingly, the minor allele T of SNP rs2043211 showed significant association with a protective effect of ischemic stroke under either a recessive model (P-obs=3.0×10–4) or an additive mode (P-obs=2.88×10–4; Table 2). After multivariate logistic regression analysis by adjusting for covariates of the age, sex, body mass index, smoking history, hypertension, diabetes mellitus, and lipid concentrations, the genotypic association between rs2043211 and ischemic stroke remained significant only under the recessive model (P-adj=0.028; OR, 0.68; Table 2).
To confirm the initial finding of genotypic association between rs2043211 and ischemic stroke in the discovery population, we validated the finding in an independent replication cohort. The results showed that rs2043211 was also significantly associated with a protective effect of ischemic stroke under either a recessive model (P-obs=9.00×10–3) or an additive mode (P-obs=4.26×10–5; Table 2). The genotypic association between rs2043211 and ischemic stroke remained significant only under the recessive model in the replication population after multivariate logistic regression analysis (P-adj=0.017; OR, 0.70; Table 2).
For the combined population of the discovery and replication cohorts, the P value for the genotypic association between rs2043211 and ischemic stroke under the recessive model became much more significant (P-obs=9.78×10–6; P-adj=5.83×10–4, OR, 0.70; Table 2; Figure I in the online-only Data Supplement). These data suggest that SNP rs2043211 confers a protective effect of ischemic stroke under a recessive model of inheritance.
The genotypic association between rs2043211 and ischemic stroke under the recessive model was more significant in the female group (P-adj=0.007; OR, 0.65) than in the male group (P-adj=0.024; OR, 0.73; Table 2; Figure I in the online-only Data Supplement). The genotypic association was significant in the early onset (<60 years) ischemic stroke group under the recessive model (P-adj=0.001; OR, 0.61; Table 2; Figure I in the online-only Data Supplement). The genotypic association was stronger under the recessive model for the female early onset ischemic stroke group (P-adj=0.004; OR, 0.51; Table 2; Figure I in the online-only Data Supplement).
Lack of Significant Association Between SNP rs2043211 and CAD
We also analyzed SNP rs2043211 for its association with CAD. In a case–control study with 772 CAD cases and 873 controls, SNP rs2043211 did not show any significant association with CAD in the standard allelic association analysis (P-obs=0.235; P-adj=0.300) or in the genotypic association analysis under 3 different genetic models (all P>0.05). The association remained nonsignificant in either male or female CAD groups (Table IV in the online-only Data Supplement).
In the present study, we identified CARD8 as a downstream gene regulated by ANRIL and demonstrated an association between the CARD8 SNP rs2043211 and ischemic stroke in 2 independent Chinese Han populations. CARD8 encodes a member of the caspase recruitment domain (CARD)–containing family and is also known as TUCAN/CARDINAL. Previous population-based studies found that the functional SNP rs2043211 (p.C10X) located in exon 5 of CARD8 may be a genetic risk factor for chronic inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis. Our finding that rs2043211 is associated with ischemic stroke could indicate a shared inflammatory response or pathway similar to inflammatory bowel disease and rheumatoid arthritis.
SNP rs2043211 has been previously associated with other diseases. Roberts et al12 observed a significant genotypic association in New Zealand between rs2043211 and abdominal aortic aneurysm (P=0.047; OR, 0.83), which is a disease that shared some similar pathological characteristics and risk factors with atherosclerosis, such as inflammation and angiogenesis. However, Paramel et al10 found that there was no significant association between rs2043211 and myocardial infarction (FIA [First Myocardial Infarction in Northern Sweden] cohort: P=0.10; OR, 1.1 and SCARF [Stockholm Coronary Atherosclerosis Risk Factor]: P=0.66; OR, 1.0). García-Bermúdez et al13 found that there was no evidence for the role of rs2043211 in the development of cardiovascular events in Spanish patients with rheumatoid arthritis (P=0.67; OR, 0.96). Meanwhile, there were still some conflicting results in association studies between rs2043211 and some chronic inflammatory diseases. Roberts et al14 found that the minor allele T of rs2043211 conferred a potential protective effect against early disease onset of Crohn disease in New Zealand whites. But other studies reported that the minor allele T of rs2043211 was associated with increased severity of inflammatory bowel disease15 (P=0.001; OR, 1.50) and rheumatoid arthritis,16 as well as increased risk of Alzheimer disease17 in women (P=0.01; OR, 2.39). Intriguingly, SNP rs2043211 was associated with ischemic stroke but not with CAD in this study. These results are consistent with the findings for myocardial infarction and cardiovascular events by Paramel et al10 and García-Bermúdez et al.13 Although the underlying molecular mechanism for the positive association with ischemic stroke and negative association with CAD by rs2043211 is not known, it is possible that the role of ANRIL-regulated CARD8 pathway may be limited to cerebral infarction but not to CAD.
CARD8 acts as an adaptor molecule that negatively regulates nuclear factor κB activation, caspase 1-dependent interleukin-1β secretion, and apoptosis and reduces the inflammatory response.18 SNP rs2043211 results in an A to T transversion that changes codon 10 into a stop codon in CARD8 mRNA (Cys10Stop). Previous studies showed that homozygotes for the stop codon allele T can reduce the expression of CARD8 and can impair the nuclear factor κB–inhibiting property of CARD8 in vitro.19 Moreover, the expression of CARD8 mRNA was significantly higher in atherosclerotic lesions from carotid artery plaque tissue in patients with ischemic cerebrovascular when compared with iliac arteries from brain dead transplant donors.10 But, more studies will be needed to investigate how CARD8 SNP rs2043211 influences the process of ischemic stroke.
These data suggest that ANRIL acts as an important modulator for expression of its downstream gene CARD8. This is consistent with the suggestion that ANRIL might modulate atherogenic diseases by cis- or trans-acting effects.6 Recently, Holdt et al20 suggested a novel role for Alu elements in epigenetic gene regulation by long ncRNAs. Importantly, transregulation was dependent on Alu motifs that marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts.20 As with other downstream target genes, the detailed mechanism by which ANRIL regulates CARD8 expression remains to be identified. We performed bioinformatic analysis of the promoter sequence of CARD8 and surprisingly found that the same core Alu motif was also present in the DNA promoter sequence of CARD8. Thus, the mechanism by which ANRIL regulates CARD8 expression may be that ANRIL binds to chromatin through interaction via the Alu motif.
There are some limitations in the present study. First, although the mRNA level of CARD8 is regulated by ANRIL, the precise underlying molecular mechanism is not clear. Second, the sample sizes are fixed and frequencies of covariates, including age, sex, smoking history, hypertension, diabetes mellitus, and lipid concentrations, are significantly lower in controls than in cases. Nevertheless, the observed association remained significant after adjustment of covariates using multiple logistic regression analysis. Third, although rs2043211 is a functional SNP, it may still serve as a genetic marker, and further studies are needed to establish the cause–effect relationship. Fourth, in addition to CARD8, our preliminary microarray analysis identified another gene, DDX58, that also showed >2-fold differential expression in HepG2 cells treated with ANRIL siRNA when compared with those transfected with control siRNA. DDX58, also referred to as RIG-1, is involved in host antivirus immunity by sensing viral RNAs and triggering innate antiviral responses.21 Because an antiviral response does not seem to be closely linked to atherosclerosis and stroke, DDX58 was not assessed for its association with stroke in this study. However, because DDX58 is regulated by ANRIL, it may still be interesting to investigate whether variants in DDX58 are associated with atherosclerosis in the future. Finally, previous studies showed that the 9p21 locus (the ANRIL locus) is associated with large-vessel atherosclerotic stroke; thus, it may be interesting to investigate whether CARD8 variant is associated with a specific stroke subtype in the future. Several schemes were reported to classify stroke subtypes, including the Trial of ORG 10172 in Acute Stroke Treatment classification system, the Causative Classification System, or the phenotypic System A-S-C-O (A for atherosclerosis, S for small vessel disease, C for cardiac source, O for other cause).22 In the present study, many patients with specific subtypes were excluded, thus we were unable to perform association studies with specific stroke subtypes. In the future, we can collect more clinical information, including the data from the carotid study, classify the cases into different subtypes, and assess the association between CARD8 SNP rs2043211 and individual stroke subtype.
We show that ANRIL can regulate the expression level of CARD8 mRNA and a functional SNP in CARD8, rs2043211 located in exon 5 of CARD8 was significantly associated with ischemic stroke but not with CAD in the Chinese Han population. Although the detailed mechanism of CARD8 in the pathogenesis of ischemic stroke remains unclear, this study indeed links the ANRIL-regulated CARD8 pathway to the development of ischemic stroke.
We thank one of the reviewers for restructuring the Abstract and for many valuable suggestions to improve the article.
Sources of Funding
This study was supported by grants from the National Basic Research Program of China (973 Program No. 2013CB0531101 and 2012CB517800), the Innovative Development of New Drugs Key Scientific Project (2011ZX09307-001-09), National Institutes of Health R01 HL094498, the Program for New Century Excellent Talents in University of China (NCET-11–0181), the Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education, the Hubei Province's Outstanding Medical Academic Leader Program, the National Natural Science Foundation of China (No. 81270163 and 81070106), and a grant from the State Key Laboratory of Freshwater Ecology and Biotechnology (2011FB16). The research at Center for Human Genome Research on lipids and coronary artery disease is supported by a collaborative grant from Merck.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.003393/-/DC1.
- Received September 4, 2013.
- Revision received November 22, 2013.
- Accepted November 27, 2013.
- © 2014 American Heart Association, Inc.
- Roger VL,
- Go AS,
- Lloyd-Jones DM,
- Benjamin EJ,
- Berry JD,
- Borden WB,
- et al
- Holdt LM,
- Teupser D
- Kastbom A,
- Johansson M,
- Verma D,
- Söderkvist P,
- Rantapää-Dahlqvist S
- Razmara M,
- Srinivasula SM,
- Wang L,
- Poyet JL,
- Geddes BJ,
- DiStefano PS,
- et al
- Marnane M,
- Duggan CA,
- Sheehan OC,
- Merwick A,
- Hannon N,
- Curtin D,
- et al