Abstract
Background
Low density lipoprotein receptor related proteins-1 and 6 have been implicated in cerebral ischemia. In addition, genetic variation in LRP1 and LRP6 has been linked with various factors that are related to risk of ischemic stroke. The aim of this study was to examine the association of LRP1 and LRP6 gene variants with risk of ischemic stroke as part of the Ischemic Stroke Genetics Study (ISGS).
Methods
We included a Caucasian series (434 stroke patients, 319 controls) and an African American series (161 stroke patients, 116 controls). Fourteen LRP6 variants and 3 LRP1 variants were genotyped and assessed for association with ischemic stroke.
Results
In the Caucasian series, significant associations with ischemic stroke were observed for LRP6 rs2075241 (OR:0.42, P=0.023), rs2302685 (OR:0.44, P=0.049), rs7975614 (OR: 0.07, P=0.017), rs10492120 (OR: 0.62, P=0.036), and rs10743980 (OR: 0.66, P=0.037). Risk of ischemic stroke was significantly lower for carriers of any of these five protective LRP6 variants (24.0% of subjects) compared to non-carriers (OR:0.57, P=0.003). The protective association for LRP6 rs2075241 was observed at a similar magnitude across ischemic stroke subtypes, while the effects of rs23022685, rs10492120, and rs10743980 were most apparent for cardioembolic and large vessel stroke. In the African American series, LRP1 rs11172113 was associated with an increased risk of stroke (OR:1.89, P=0.006).
Conclusions
The results of our preliminary study provide evidence that LRP6 and LRP1 variants may be associated with risk of ischemic stroke. Validation in larger studies is warranted.
Keywords: genetics, ischemic stroke, low density lipoprotein receptor related protein
1.1 Introduction
Stroke is a leading cause of disability and death throughout the world, occurring in 795,000 people each year in the United States alone and causing approximately 1 of every 19 deaths[1]. Clinical and environmental risk factors for ischemic stroke, such as older age, hypertension, high cholesterol, and smoking have been well documented. Additionally, a number of recent advances have been made regarding the genetics of ischemic stroke[2]. For example, a meta-analysis of genome-wide association studies (GWAS) involving 12,389 ischemic stroke patients and 62,004 controls confirmed associations of PITX2 and ZFHX2 with cardioembolic stroke and associations of HDAC9 and the chromosome 9p21 locus with risk of large-vessel stroke[3]. However, despite these important findings, much remains to be understood regarding genetic causes of ischemic stroke, particularly in African Americans who have a high risk of ischemic stroke and who have been poorly studied regarding ischemic stroke genetics.
Low density lipoprotein receptor related protein (LRP) signaling is involved in multiple brain processes, including neuronal excitation, cerebrovascular remodeling, and cerebral ischemia. LRP1, which is highly expressed in neurons, binds multiple ligands, and mediates vesicle and transmembrane transport, synaptic function, and brain metabolism[4],[5]. LRP1 is additionally involved in vascular homeostasis and can influence smooth muscle cell proliferation, vascular inflammatory markers and atherosclerosis[6-8]. Evidence of LRP involvement in cerebral ischemia also comes from animal studies. For example, penumbral LRP1 expression increases following experimental middle cerebral artery occlusion (MCAO), while nonspecific LRP-antagonists increase return of function following MCAO in animal models[9, 10]. Likewise, LRP6 haploinsufficiency increases proinflammatory markers, mitochondrial dysfunction and stroke volume[11]. Additionally, genetic variants in LRP1 and LRP6 have been associated with various measures that are related to ischemic stroke, such as risk of migraine, risk of abdominal aortic aneurysm, effectiveness of statins in reducing risk of myocardial infarction, and LDL cholesterol[12-16]. Taken together, these findings raise the possibility that LRP gene variants may play a role in determining risk of ischemic stroke. Therefore, in this preliminary investigation we evaluated LRP1 and LRP6 variants for association with risk of ischemic stroke and ischemic stroke subtypes in Caucasians and African Americans.
2.1 Methods
2.1.1 Study subjects
A total of 595 ischemic stroke patients and 435 controls were included in this study. These individuals were from an Ischemic Stroke Genetics Study (ISGS) Caucasian series (434 patients, 319 controls) and an ISGS African American series (161 patients, 116 controls). All study participants gave written informed consent for participation in this study and approval obtained from the relevant institutional ethics committees. Information was collected for all individuals regarding age, gender, atrial fibrillation, coronary artery disease, diabetes, hypertension, and smoking. Type of stroke (cardioembolic, large vessel, small vessel, other, undetermined) was also collected for ischemic stroke patients. Stroke was defined by the World Health Organization criteria as rapidly developing signs of a focal or global disturbance of cerebral function, with symptoms lasting at least 24 hours or leading to death, with no apparent cause other than vascular origin[17]. Stroke was classified as an ischemic stroke when magnetic resonance imaging or computed tomography of the brain was performed within 7 days of stroke symptom onset and identified the symptomatic cerebral infarct or failed to identify an alternative cause of symptoms. Ischemic stroke subtypes were classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) system[18]. A summary of subject characteristics is provided in Table 1 for the ISGS Caucasian and ISGS African American series.
Table 1. Patient characteristics in the ISGS Caucasian and ISGS African American series.
| ISGS Caucasian series | ISGS African American series | |||
|---|---|---|---|---|
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| ||||
| Variable | Stroke patients (N=434) | Controls (N=319) | Stroke patients (N=161) | Controls (N=116) |
| Age | 65 ± 14 (22 – 94) | 62 ± 15 (22 – 92) | 55 ± 13 (19 – 87) | 53 ± 13 (22 – 90) |
| Gender (Male) | 256 (59%) | 157 (49%) | 80 (50%) | 46 (40%) |
| Atrial fibrillation | 32 (7%) | 8 (3%) | 8 (5%) | 0 (0%) |
| Coronary artery disease | 118 (27%) | 35 (11%) | 17 (11%) | 7 (6%) |
| Diabetes | 101 (23%) | 34 (11%) | 50 (31%) | 26 (22%) |
| Hypertension | 284 (65%) | 118 (37%) | 131 (81%) | 50 (44%) |
| Current smoking | 97 (22%) | 22 (7%) | 59 (37%) | 18 (16%) |
| Type of stroke | ||||
| Cardioembolic | 121 (28%) | N/A | 28 (17%) | N/A |
| Large-vessel | 86 (20%) | N/A | 27 (17%) | N/A |
| Small-vessel | 60 (14%) | N/A | 43 (27%) | N/A |
| Other | 24 (6%) | N/A | 2 (1%) | N/A |
| Undetermined | 143 (33%) | N/A | 61 (38%) | N/A |
The sample mean ± SD (minimum – maximum) is given for age. In the ISGS Caucasian series, information was unavailable regarding atrial fibrillation (N=3), coronary artery disease (N=1), and hypertension (N=1). In the ISGS African American series, information was unavailable for atrial fibrillation (N=4), coronary artery disease (N=1), and hypertension (N=2). ISGS=Ischemic Stroke Genetics Study. SD=standard deviation.
2.1.2 Genetic analysis
We selected 3 LRP1 variants and 14 LRP6 variants for analysis in this study (Table 2). LRP1 variants were selected due to previously demonstrated associations with migraine, abdominal aortic aneurysm, or myocardial infarction[12-14, 16]. Twelve of the 14 LRP6 variants were common tagging variants previously studied by Tomaszewski et al. in relation to LDL-cholesterol[15]. The rs121918313 LRP6 variant was included due to an association with coronary artery disease and metabolic syndrome[19, 20], and the LRP6 variant rs7975614 was included because it was identified as the only other common non-synonymous variant in the gene in African Americans based on data from the Exome Variant Server (minor allele frequency 17.7%). Details of these variants and their reasons for inclusion are provided in Table 2.
Table 2. Variant information and reason for inclusion.
| SNP information | Reason for inclusion in current study | ||||
|---|---|---|---|---|---|
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| Variant | Chromosome | Position (bp)a | Gene | Disease or outcome of interest previously studied in: | Reference(s) |
| rs12313200 | 12p13 | 12284590 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs2075241 | 12p13 | 12291479 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs7980903 | 12p13 | 12291638 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs11054704 | 12p13 | 12299769 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs2302685 | 12p13 | 12301898 | LRP6 | LDL-cholesterol LDL-cholesterol |
Tomaszewski et al 2009 |
| rs11609634 | 12p13 | 12309687 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs121918313 | 12p13 | 12317428 | LRP6 | Metabolic syndrome | Mani et al 2007 |
| rs2417085 | 12p13 | 12331375 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs10845493 | 12p13 | 12332196 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs7975614 | 12p13 | 12332842 | LRP6 | Common non-synonymous variant in African Americans | Exome Variant Server data |
| rs10492120 | 12p13 | 12333352 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs17302049 | 12p13 | 12365325 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs11054738 | 12p13 | 12388027 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs10743980 | 12p13 | 12412795 | LRP6 | LDL-cholesterol | Tomaszewski et al 2009 |
| rs11172113 | 12q13 | 57527283 | LRP1 | Migraine | Chasman et al 2011, Freilinger et al 2012 |
| rs715948 | 12q13 | 57532982 | LRP1 | Effectiveness of statins in reducing risk of myocardial infarction | Peters et al 2011 |
| rs1466535 | 12q13 | 57534470 | LRP1 | Abdominal aortic aneurysm | Bown et al 2011 |
Chromosomal positions based on the February 2009 (GRCH37/hg19) genome assembly. MAF = Minor allele frequency
DNA was extracted from whole blood samples using standard protocols. Variants were genotyped by TaqMan Allelic Discrimination Assay on an ABI 7900HT Fast Real-Time PCR system (Life Technologies, Foster City, CA) using manufacturer protocol and analyzed using SDS v2.3 software (Life Technologies). Genotype call rates were >95% for each variant for each series. There was no evidence of a departure from Hardy Weinberg Equilibrium for any variants (all P<0.05 after Bonferroni correction) except rs7975614 in the ISGS Caucasian series which was due to one rare homozygote; genotypes for this variant were checked for accuracy and retained for use in analysis. Linkage disequilibrium (LD) between LRP6 variants and LRP1 variants in controls was assessed using r-squared values; summaries of LD are displayed in Supplemental Figures 1a-1d for each series and each gene. Of note, no ischemic stroke cases or controls had a copy of the minor allele for LRP6 rs121918313; r-squared is undefined for variant combinations involving this variant. Therefore, although this variant is included in Figures 1a-1d, no r-squared values are given.
2.1.3 Statistical analysis
All analysis was performed separately for the ISGS Caucasian series and the ISGS African American series. Associations of LRP6 and LRP1 variants with risk of ischemic stroke and ischemic stroke subtypes (cardioembolic, large-vessel, small-vessel) were evaluated using logistic regression models adjusted for age, gender, atrial fibrillation, coronary artery disease, diabetes, hypertension, and smoking. Atrial fibrillation was not adjusted in the ISGS African American series owing to the absence of controls with atrial fibrillation in that series. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated. We considered genotypes for each variant under an additive model (effect of each additional minor allele), dominant model (presence vs. absence of the minor allele), and recessive model (presence vs. absence of two copies of the minor allele). For associations of LRP6 and LRP1 variants with risk of ischemic stroke subtypes, these were not examined in the ISGS African American series owing to its small sample size. No adjustment for multiple testing was made in these exploratory analyses, and p-values of 0.05 or less were considered as statistically significant. All statistical analysis was performed using SAS (version 9.2; SAS Institute, Inc., Cary, North Carolina) and R Statistical Software (version 2.14.0; R Foundation for Statistical Computing, Vienna, Austria).
3.1 Results
An evaluation of associations of LRP6 and LRP1 variants with risk of ischemic stroke in the ISGS Caucasian series is shown in Table 3. Significant associations with ischemic stroke were observed for LRP6 rs2075241 under a recessive model (OR: 0.42, P=0.023), LRP6 rs2302685 under a recessive model (OR: 0.44, P=0.049), LRP6 rs7975614 under an additive (OR: 0.08: P=0.021) and a dominant model (OR: 0.07, P=0.017), LRP6 rs10492120 under a recessive model (OR: 0.62, P=0.036), and LRP6 rs10743980 under a recessive model (OR: 0.66, P=0.037). The latter two variants (rs10743980 and rs10492120) are in linkage disequilibrium with an r-squared of 0.77. The very strong odds ratio for the rare LRP6 rs7975614 variant is due to the fact that only 1 ischemic stroke patient (0.2%) had a copy of the minor allele, compared to 8 controls (2.5%). Given that the associations for each of the five aforementioned were protective and also involved rare genotypes, we collapsed across these five variants and examined the association between presence of any of the five protective genotypes and ischemic stroke risk; in the carriers of a protective genotype for at least one of these five LRP6 variants (24.0% of subjects), risk of ischemic stroke was significantly lower (OR: 0.57, 95% CI: 0.39 – 0.82, P=0.003) compared to non-carriers. There were no other significant associations between LRP6 variants and risk of ischemic stroke in the ISGS Caucasian series (all P≥0.060, Table 2), and none of the three LRP1 variants showed evidence of an association with ischemic stroke in this series (all P≥0.50, Table 2).
Table 3. Associations of LRP6 and LRP1 variants with risk of ischemic stroke in the ISGS Caucasian series.
| Additive model | Dominant model | Recessive model | |||||||
|---|---|---|---|---|---|---|---|---|---|
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| |||||||||
| Variant | Gene | MA | MAF | OR (95% CI) | P-value | OR (95% CI) | P-value | OR (95% CI) | P-value |
| rs12313200 | LRP6 | T | 15.5% | 1.23 (0.89, 1.69) | 0.21 | 1.15 (0.81, 1.65) | 0.43 | 3.04 (0.95, 9.71) | 0.060 |
| rs2075241 | LRP6 | G | 18.5% | 0.80 (0.61, 1.05) | 0.11 | 0.85 (0.61, 1.20) | 0.37 | 0.42 (0.20, 0.89) | 0.023 |
| rs7980903 | LRP6 | G | 18.2% | 1.05 (0.78, 1.40) | 0.77 | 1.14 (0.81, 1.60) | 0.46 | 0.65 (0.28, 1.51) | 0.31 |
| rs11054704 | LRP6 | T | 14.8% | 1.14 (0.83, 1.57) | 0.41 | 1.24 (0.86, 1.77) | 0.25 | 0.72 (0.26, 2.02) | 0.54 |
| rs2302685 | LRP6 | C | 19.0% | 0.83 (0.63, 1.10) | 0.19 | 0.88 (0.63, 1.24) | 0.47 | 0.44 (0.20, 1.00) | 0.049 |
| rs11609634 | LRP6 | T | 47.1% | 1.09 (0.88, 1.35) | 0.45 | 1.19 (0.84, 1.69) | 0.32 | 1.05 (0.72, 1.52) | 0.80 |
| rs121918313 | LRP6 | --- | --- | --- | --- | --- | --- | --- | --- |
| rs2417085 | LRP6 | C | 46.0% | 0.91 (0.72, 1.13) | 0.38 | 1.00 (0.70, 1.41) | 0.98 | 0.74 (0.50, 1.10) | 0.13 |
| rs10845493 | LRP6 | A | 14.5% | 1.16 (0.84, 1.60) | 0.36 | 1.26 (0.88, 1.81) | 0.21 | 0.73 (0.26, 2.03) | 0.54 |
| rs7975614 | LRP6 | A | 0.7% | 0.08 (0.01, 0.68) | 0.021 | 0.07 (0.01, 0.63) | 0.017 | N/A | N/A |
| rs10492120 | LRP6 | G | 37.7% | 0.84 (0.67, 1.05) | 0.13 | 0.90 (0.65, 1.24) | 0.52 | 0.62 (0.40, 0.97) | 0.036 |
| rs17302049 | LRP6 | C | 14.3% | 1.23 (0.88, 1.70) | 0.23 | 1.18 (0.82, 1.70) | 0.37 | 2.74 (0.70, 10.72) | 0.15 |
| rs11054738 | LRP6 | T | 18.3% | 1.05 (0.78, 1.40) | 0.76 | 1.10 (0.78, 1.54) | 0.60 | 0.84 (0.36, 1.95) | 0.68 |
| rs10743980 | LRP6 | A | 44.1% | 0.88 (0.70, 1.09) | 0.23 | 0.99 (0.71, 1.39) | 0.97 | 0.66 (0.45, 0.98) | 0.037 |
| rs11172113 | LRP1 | C | 37.3% | 0.99 (0.78, 1.24) | 0.90 | 1.00 (0.72, 1.38) | 0.99 | 0.95 (0.60, 1.50) | 0.82 |
| rs715948 | LRP1 | T | 31.3% | 0.99 (0.77, 1.26) | 0.91 | 1.04 (0.76, 1.43) | 0.80 | 0.83 (0.48, 1.43) | 0.50 |
| rs1466535 | LRP1 | A | 31.6% | 1.05 (0.82, 1.33) | 0.72 | 1.06 (0.77, 1.46) | 0.73 | 1.06 (0.63, 1.79) | 0.84 |
ORs and p-values result from logistic regression models adjusted for age, gender, atrial fibrillation, coronary artery disease, diabetes, hypertension, and current smoking. ORs correspond to an additional minor allele (additive models), presence of the minor allele (dominant models), or presence of two copies of the minor allele (recessive models). --- indicates a variant for which the minor allele was not observed. N/A indicates a variant for which either no patient or control had two copies of the minor allele, making analysis under a recessive model impossible. MA=minor allele. MAF=minor allele frequency. OR=odds ratio. CI=confidence interval. ISGS=Ischemic Stroke Genetics Study.
Associations of LRP6 and LRP1 variants with risk of ischemic stroke in the ISGS African American series are displayed in Table 4. The only variant that was associated with stroke in the ISGS African American series was LRP1 rs11172113, where significant associations were observed under an additive model (OR: 1.89, P=0.006), a dominant model (OR: 2.18, P=0.019), and a recessive model (OR: 2.24, P=0.047). There were no other significant associations with risk of ischemic stroke for LRP6 and LRP1 variants in the ISGS African American series (all P≥0.10, Table 3).
Table 4. Associations of LRP6 and LRP1 variants with risk of ischemic stroke in the ISGS African American series.
| Additive model | Dominant model | Recessive model | |||||||
|---|---|---|---|---|---|---|---|---|---|
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| Variant | Gene | MA | MAF | OR (95% CI) | P-value | OR (95% CI) | P-value | OR (95% CI) | P-value |
| rs12313200 | LRP6 | T | 9.0% | 0.72 (0.35, 1.47) | 0.37 | 0.72 (0.35, 1.47) | 0.37 | N/A | N/A |
| rs2075241 | LRP6 | G | 25.6% | 0.99 (0.63, 1.56) | 0.96 | 0.83 (0.47, 1.46) | 0.52 | 2.06 (0.61, 6.95) | 0.24 |
| rs7980903 | LRP6 | G | 12.8% | 1.11 (0.62, 2.01) | 0.72 | 1.12 (0.58, 2.16) | 0.74 | 1.23 (0.14, 11.02) | 0.86 |
| rs11054704 | LRP6 | T | 7.6% | 1.67 (0.80, 3.48) | 0.17 | 2.01 (0.87, 4.65) | 0.10 | 0.48 (0.02, 11.1) | 0.65 |
| rs2302685 | LRP6 | C | 11.6% | 1.39 (0.74, 2.63) | 0.31 | 1.54 (0.78, 3.04) | 0.22 | N/A | N/A |
| rs11609634 | LRP6 | T | 18.4% | 1.00 (0.62, 1.61) | 0.99 | 1.04 (0.56, 1.94) | 0.91 | 0.86 (0.27, 2.76) | 0.80 |
| rs121918313 | LRP6 | --- | --- | --- | --- | --- | --- | --- | --- |
| rs2417085 | LRP6 | T | 19.8% | 0.82 (0.50, 1.34) | 0.43 | 0.90 (0.27, 3.01) | 0.86 | 0.75 (0.40, 1.39) | 0.36 |
| rs10845493 | LRP6 | A | 2 4% | 1.58 (0.58, 4.25) | 0.37 | 1.87 (0.60, 5.87) | 0.28 | 0.67 (0.02, 23.23) | 0.83 |
| rs7975614 | LRP6 | A | 15.5% | 1.01 (0.58, 1.75) | 0.98 | 0.98 (0.53, 1.79) | 0.94 | 1.47 (0.19, 11.24) | 0.71 |
| rs10492120 | LRP6 | A | 48.7% | 0.93 (0.63, 1.37) | 0.71 | 0.81 (0.43, 1.54) | 0.52 | 1.01 (0.54, 1.90) | 0.98 |
| rs17302049 | LRP6 | C | 4.3% | 0.82 (0.30, 2.26) | 0.71 | 0.82 (0.30, 2.26) | 0.71 | N/A | N/A |
| rs11054738 | LRP6 | T | 9.1% | 0.66 (0.33, 1.32) | 0.24 | 0.64 (0.30, 1.34) | 0.23 | 0.66 (0.02, 22.25) | 0.82 |
| rs10743980 | LRP6 | G | 41.2% | 1.23 (0.83, 1.80) | 0.30 | 1.06 (0.53, 2.14) | 0.87 | 1.54 (0.85, 2.77) | 0.15 |
| rs11172113 | LRP1 | C | 44.8% | 1.89 (1.20, 2.99) | 0.006 | 2.18 (1.14, 4.18) | 0.019 | 2.24 (1.01, 4.96) | 0.047 |
| rs715948 | LRP1 | T | 22.7% | 0.71 (0.43, 1.17) | 0.18 | 0.77 (0.43, 1.35) | 0.36 | 0.23 (0.04, 1.36) | 0.11 |
| rs1466535 | LRP1 | A | 22.9% | 1.07 (0.67, 1.69) | 0.78 | 1.03 (0.59, 1.80) | 0.93 | 1.39 (0.41, 4.78) | 0.60 |
ORs and p-values result from logistic regression models adjusted for age, gender, coronary artery disease, diabetes, hypertension, and current smoking. ORs correspond to an additional minor allele (additive models), presence of the minor allele (dominant models), or presence of two copies of the minor allele (recessive models). --- indicates a variant for which the minor allele was not observed. N/A indicates a variant for which either no patient or control had two copies of the minor allele, making analysis under a recessive model impossible. MA=minor allele. MAF=minor allele frequency. OR=odds ratio. CI=confidence interval. ISGS=Ischemic Stroke Genetics Study.
LRP6 and LRP1 variants were examined in relation to risk of stroke subtypes in the ISGS Caucasian series, and these results are displayed in Supplemental Table 1 (additive model), Supplemental Table 2 (dominant model), and Supplemental Table 3 (recessive model). There were no statistically significant associations between LRP6 or LRP1 variants and stroke subtypes. Of note, although not approaching statistical significance in this lower-powered analysis, the aforementioned protective association for LRP6 rs2075241 was observed at a similar magnitude across ischemic stroke subtypes, whereas the protective effects of rs23022685, rs10492120, and rs10743980 were most apparent for cardioembolic and large vessel stroke. Ischemic stroke subtype analysis was not performed for LRP6 rs7975614 due to its low frequency. A detailed summary of allele and genotype frequencies in stroke patients and controls is shown in Supplemental Tables 4a-4f for both the Caucasian and African American series.
4.1 Discussion
To date, much of the success in identifying genetic risk factors for ischemic stroke has come from studying genes that are associated with diseases that have links to stroke, such as coronary artery disease and myocardial infarction (chromosome 9p21) as well as atrial fibrillation (PITX2, ZFHX3). The involvement of LRP1 and LRP6 in cerebral ischemia, combined with previously demonstrated associations with stroke-linked factors such as LDL cholesterol, abdominal aortic aneurysm, and migraine, suggest that LRP1 and LRP6 are promising candidates for study as potential genetic risk factors for ischemic stroke. The results of the current study provide evidence that genetic variants in LRP1 and LRP6 genes may be associated with ischemic stroke. Importantly, the associations with ischemic stroke that we observed were independent of traditional risk factors for ischemic stroke such as coronary artery disease, diabetes, hypertension, and smoking. Although it is very important to highlight that these findings should be viewed as preliminary due to the relatively small sample size, they are promising nonetheless.
In our Caucasian series, we identified several LRP6 variants that had significant protective associations for ischemic stroke risk in Caucasians. Associations between LRP6 and ischemic stroke have not been identified in previous GWAS's, however this may not be surprising given that the associations observed in our study were either observed under a recessive model that is not utilized in GWAS's (rs2075241, rs2302685, rs10492120, rs10743980) or involved rare variants that would likely not have been included in a GWAS (rs7975614). Furthermore, risk of ischemic stroke was decreased more than 1.7-fold in subjects who were carriers of the protective genotype for any of the five aforementioned LRP6 variants (P=0.003). We additionally identified a LRP1 variant (rs11172113) that may be associated with risk of ischemic stroke in African Americans. Although the association for this variant was the strongest that we observed in our study with an odds ratio of 1.89 and p-value of 0.006 under an additive model, it should be interpreted with caution due to the small size of our African American series. Additionally, stroke subtype analysis was not performed in the small African American series, however based on a simple examination of allele frequencies, this association appears to have been driven mostly by large vessel and small-vessel stroke, where minor allele frequencies were higher (51.9% and 47.7%, respectively) compared to controls (39.7%) and cardioembolic stroke (42.9%). Further study is needed.
Several limitations of our study should be noted. Due to the aforementioned relatively small sample size, power to detect associations of LRP6 and LRP1 variants with risk of ischemic stroke is low, and therefore the possibility of type II error (i.e. a false-negative association) is important to consider. This is especially true in the small African American series, for variants with lower minor allele frequencies, and when evaluating associations with ischemic stroke subtypes. Related to this, LRP6 rs7975614, which was significantly associated with ischemic stroke in the Caucasian series, was very rare with a minor allele frequency of 0.7% in that series. Due to this low frequency, estimates of the association between rs7975614 and risk of ischemic stroke are very imprecise, and examination of this variant in a larger series will be important. Additionally, due to the relatively small sample size and corresponding exploratory nature of this study, we made no adjustment for multiple testing despite the relatively large number of statistical tests that were performed. Although this strategy lowers the likelihood of a type II error, it increases the probability of obtaining a type I error (i.e. a false-positive association), and therefore it is important to highlight the need for validation of our findings. Of note, we made an effort to perform such a validation of our findings in Caucasians by utilizing the Metastroke database[21], which is a collaborative effort of combined data from 15 different GWAS studies including individuals of European ancestry. However, we observed almost all of our significant findings in Caucasians under a recessive genotypic model, and because results under this model were not available in the Metastroke database, we were unable to present these validation results herein. Also, of the 12 LRP6 variants that were previously studied by Tomaszewski et al. in relation to LDL-cholesterol and therefore included in this study, only 1 variant (rs10845493) was significant associated with LDL-cholesterol[15], however this was not replicated in a recent very large study[22] and therefore a role for this LRP6 variant in modifying LDL-cholesterol is unlikely. Although LRP6 variants were not identified in these recent GWA studies[23, 24], the authors show that those identified explain only a small fraction of the variation in circulating lipid levels and likely do not account for all of the variations associated with these traits. Finally, it should be mentioned that the results of many candidate gene studies such as this one have failed to replicate, which again points to the need for validation of our findings.
Acknowledging these limitations, our preliminary results indicate that genetic variants in LRP6 and LRP1 may play a role in determining risk of ischemic stroke. Given that genetic risk factors for ischemic stroke appear to be subtype-specific, larger studies are needed to better examine how these variants (whose significant associations were mostly driven by relatively rare genotypes) may affect risk of specific ischemic stroke subtypes. Additionally, re-examination of previously published GWAS data for LRP6 rs2075241, rs2302685, rs10492120, and rs10743980 under a recessive rather than additive model appears to be warranted, and it will also be of interest to build on our findings involving LRP1 rs11172113 in other series of African American individuals.
Supplementary Material
Acknowledgments
OAR is a recipient of a James and Ester King Foundation New Investigator Award from the Department of Health, Florida State and funded by the American Heart Association (AHA) and the Myron and Jane Hanley Award in Stroke research. The Siblings with Ischemic Stroke Study was funded by a grant from the NINDS (R01 NS39987; JFM, PI) and by a Marriott Disease Risk and Regenerative Medicine Initiative Award in Individualized Medicine. The Ischemic Stroke Genetics Study (ISGS) was funded by a grant from the NINDS (R01 NS42733). ZKW is partially supported by the NIH/NINDS NS057567, P50 NS072187. For further details on our research in cerebrovascular disease please visit our web-site; http://mayoresearch.mayo.edu/mayo/research/ross_lab/.
Abbreviations
- LRP
Low density lipoprotein receptor related protein
- ISGS
Ischemic Stroke Genetics Study
- GWAS
Genome wide association studies
Footnotes
The authors declare no financial or other conflict of interests.
References
- 1.Go AS, Mozaffarian D, Roger VL, et al. Executive summary: heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation. 2014;129:399–410. doi: 10.1161/01.cir.0000442015.53336.12. [DOI] [PubMed] [Google Scholar]
- 2.Sharma P, Yadav S, Meschia JF. Genetics of ischaemic stroke. Journal of neurology, neurosurgery, and psychiatry. 2013;84:1302–1308. doi: 10.1136/jnnp-2012-304834. [DOI] [PubMed] [Google Scholar]
- 3.Traylor M, Farrall M, Holliday EG, et al. Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies. Lancet neurology. 2012;11:951–962. doi: 10.1016/S1474-4422(12)70234-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Fuentealba RA, Liu Q, Kanekiyo T, Zhang J, Bu G. Low density lipoprotein receptor-related protein 1 promotes anti-apoptotic signaling in neurons by activating Akt survival pathway. The Journal of biological chemistry. 2009;284:34045–34053. doi: 10.1074/jbc.M109.021030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Liu Q, Trotter J, Zhang J, et al. Neuronal LRP1 knockout in adult mice leads to impaired brain lipid metabolism and progressive, age-dependent synapse loss and neurodegeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010;30:17068–17078. doi: 10.1523/JNEUROSCI.4067-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK. LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. Physiological reviews. 2008;88:887–918. doi: 10.1152/physrev.00033.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Muratoglu SC, Belgrave S, Hampton B, et al. LRP1 Protects the Vasculature by Regulating Levels of Connective Tissue Growth Factor and HtrA1. Arteriosclerosis, thrombosis, and vascular biology. 2013;33:2137–2146. doi: 10.1161/ATVBAHA.113.301893. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Hopkins PN. Molecular biology of atherosclerosis. Physiological reviews. 2013;93:1317–1542. doi: 10.1152/physrev.00004.2012. [DOI] [PubMed] [Google Scholar]
- 9.Gutierrez-Fernandez M, Rodriguez-Frutos B, Fuentes B, et al. CDP-choline treatment induces brain plasticity markers expression in experimental animal stroke. Neurochemistry international. 2012;60:310–317. doi: 10.1016/j.neuint.2011.12.015. [DOI] [PubMed] [Google Scholar]
- 10.Polavarapu R, Gongora MC, Yi H, et al. Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit. Blood. 2007;109:3270–3278. doi: 10.1182/blood-2006-08-043125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Abe T, Zhou P, Jackman K, et al. Lipoprotein receptor-related protein-6 protects the brain from ischemic injury. Stroke; a journal of cerebral circulation. 2013;44:2284–2291. doi: 10.1161/STROKEAHA.113.001320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Chasman DI, Schurks M, Anttila V, et al. Genome-wide association study reveals three susceptibility loci for common migraine in the general population. Nature genetics. 2011;43:695–698. doi: 10.1038/ng.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Freilinger T, Anttila V, de Vries B, et al. Genome-wide association analysis identifies susceptibility loci for migraine without aura. Nature genetics. 2012;44:777–782. doi: 10.1038/ng.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Peters BJ, Pett H, Klungel OH, et al. Genetic variability within the cholesterol lowering pathway and the effectiveness of statins in reducing the risk of MI. Atherosclerosis. 2011;217:458–464. doi: 10.1016/j.atherosclerosis.2011.06.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Tomaszewski M, Charchar FJ, Barnes T, et al. A common variant in low-density lipoprotein receptor-related protein 6 gene (LRP6) is associated with LDL-cholesterol. Arteriosclerosis, thrombosis, and vascular biology. 2009;29:1316–1321. doi: 10.1161/ATVBAHA.109.185355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Bown MJ, Jones GT, Harrison SC, et al. Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1. American journal of human genetics. 2011;89:619–627. doi: 10.1016/j.ajhg.2011.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.The World Health Organization MONICA Project (monitoring trends and determinants in cardiovascular disease): a major international collaboration. WHO MONICA Project Principal Investigators. Journal of clinical epidemiology. 1988;41:105–114. doi: 10.1016/0895-4356(88)90084-4. [DOI] [PubMed] [Google Scholar]
- 18.Adams HP, Jr, Bendixen BH, Kappelle LJ, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke; a journal of cerebral circulation. 1993;24:35–41. doi: 10.1161/01.str.24.1.35. [DOI] [PubMed] [Google Scholar]
- 19.Mani A, Radhakrishnan J, Wang H, et al. LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science. 2007;315:1278–1282. doi: 10.1126/science.1136370. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Singh R, Smith E, Fathzadeh M, et al. Rare nonconservative LRP6 mutations are associated with metabolic syndrome. Human mutation. 2013;34:1221–1225. doi: 10.1002/humu.22360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Traylor M, Farrall M, Holliday EG, et al. Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies. Lancet Neurol. 2012;11:951–962. doi: 10.1016/S1474-4422(12)70234-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Willer CJ, Schmidt EM, Sengupta S, et al. Discovery and refinement of loci associated with lipid levels. Nature genetics. 2013;45:1274–1283. doi: 10.1038/ng.2797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Waterworth DM, Ricketts SL, Song K, et al. Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology. 2010;30:2264–2276. doi: 10.1161/ATVBAHA.109.201020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Willer CJ, Sanna S, Jackson AU, et al. Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nature genetics. 2008;40:161–169. doi: 10.1038/ng.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
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