Article
Endothelin-1 Gene Polymorphisms Influence Cerebrospinal Fluid Endothelin-1 Levels Following Aneurysmal Subarachnoid Hemorrhage
Biological Research for Nursing 2015, Vol. 17(2) 185-190 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1099800414536261 brn.sagepub.com
Matthew J. Gallek, PhD, RN1, Sheila A. Alexander, PhD, RN2, Elizabeth Crago, PhD, RN2, Paula R. Sherwood, PhD, RN2, Megan Klamerus, MS3, Michael B. Horowitz, MD4, Samuel M. Poloyac, PharmD, PhD5, and Yvette Conley, PhD2
Abstract Aneurysmal subarachnoid hemorrhage is a type of stroke with high morbidity and mortality. Increased endothelin-1 (ET-1) levels have been associated with increased risk of cerebral vasospasm, which is associated with increased morbidity. The purpose of this study was to investigate the relationships between ET-1 genotypes and ET-1 protein levels in cerebrospinal fluid (CSF) measured 72 hr before angiographic vasospasm measurement in subjects at high risk of cerebral vasospasm. Specifically, this study evaluated the differences between variant positive and variant negative groups of nine different ET-1 single-nucleotide polymorphisms (SNPs) in relationship with the ET-1 protein exposure rate. The CSF ET-1 protein levels were quantified using enzyme-linked immunosorbent assay. One functional SNP and eight ET-1 tagging SNPs were selected because they represent genetic variability in the entire ET-1 gene. The variant negative group of SNP rs2070699 was associated with a significantly higher ET-1 exposure rate than the variant positive group (p ¼ 0.004), while the variant positive group of the rs5370 group showed a trend toward association with a higher ET-1 exposure rate (p ¼ 0.051). Other SNPs were not informative. This is the first study to show differences in ET-1 exposure rate 72 hr before angiography in relation to ET-1 genotypes. These exploratory findings need to be replicated in a larger study; if replicated, these differences in genotypes may be a way to inform clinicians of those patients at a higher risk of increased ET-1 protein levels, which may lead to a higher risk of angiographic vasospasm. Keywords subarachnoid hemorrhage, endothelin-1, genetics
Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating type of stroke that occurs most often when a cerebral aneurysm ruptures. It affects nearly 30,000 people a year and has a mortality rate of approximately 30% (Nieuwkamp et al., 2009). A major cause of morbidity in this population is delayed cerebral ischemia related to cerebral vasospasm. Angiographic vasospasm occurs in up to 70% of aSAH patients (Dorsch, 2002). Endothelin (ET), a potent vasoconstrictor, is hypothesized to contribute to cerebral vasospasm. Research has shown an association between increased cerebrospinal fluid (CSF) ET-1 levels and angiographic vasospasm (Kessler, Pacheco, Lozzi, de Araujo, & Onishi, 2005; Mascia et al., 2001; Thampatty et al., 2011). Further research has shown that polymorphisms of the ET-1 gene influence levels of the ET-1 protein, but these studies have only included hypertensive patients without aSAH and measured ET-1 only in plasma (Barden et al., 2001; Tanaka,
Kamide, Takiuchi, Kawano, & Miyata, 2004). The purpose of the present study was to investigate the relationships between ET-1 protein levels in CSF measured 72 hr before angiographic vasospasm measurement and ET-1 genotypes in our sample of aSAH patients.
1
College of Nursing, University of Arizona, Tucson, AZ, USA School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA 3 Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA 4 Pennsylvania Brain and Spine Institute, Allegheny Health Network, Pittsburgh, PA, USA 5 School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA 2
Corresponding Author: Matthew J. Gallek, PhD, RN, The University of Arizona, College of Nursing, 1305 N Martin Ave, PO Box 210203, Tucson, AZ 85721, USA. Email: [email protected]
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Biological Research for Nursing 17(2) Concentration of ET-1 protein in the sample (pg/ml) CSF drainage (ml)/Time period (hr)
Material and Method Participants Participants were a subset of individuals who were recruited as part of an ongoing National Institutes of Health–funded study (R01NR004339) following approval by the University of Pittsburgh Institutional Review Board and after obtaining informed consent from family members or, in rare cases, participants themselves. Participants were recruited by hospital staff referral from the neurovascular intensive care unit (ICU) at the University of Pittsburgh Medical Center from September 2000 to April 2006. Inclusion criteria for the parent study were (1) adult patients with the diagnosis of SAH with an aneurysm verified by computerized tomographic angiography or digital subtraction angiography and (2) a Fisher grade > 1. For the present study participants must also have had a ventriculostomy or lumbar drain for CSF collection, angiography for cerebral vasospasm detection, and be non-Hispanic Whites. Although patients of all ethnic backgrounds were included in the recruiting process for the parent study, we limited analyses of our data for the present study to non-Hispanic Whites to address population stratification, given that representation of other races and ethnicities were not large enough to permit separate analyses for those groups. Exclusion criteria included (1) preexisting neurological disorder, (2) traumatic SAH, (3) mycotic aneurysm, or (4) arteriovenous malformation. All participants received standard medical care in the ICU, including triple-H therapy (hypertension, hypervolemia, and hemodilution), nimodipine, and early intervention to repair the aneurysm.
Genotyping Whole blood (3 cc) was collected from the participants within 48 hr of admission for DNA extraction using a simple salting out procedure (Miller, Dykes, & Polesky, 1988). Genotyping of the ET-1 single-nucleotide polymorphisms (SNPs) was completed using the ABI Prism1 7000 Sequence Detection System (Applied Biosystems, USA) and TaqMan1 allele discrimination assays (Applied Biosystems, USA). We chose the rs5370 SNP because it is a functional SNP that has been associated with other diseases. It is a G-to-T transversion at Position 5665 affecting the 61st nucleotide of Exon 5, corresponding with a Lys/Asn change at Codon 198. The remaining eight SNPs we genotyped were tagging SNPs and as such represent known variability within the entire gene. We consulted the HapMap.org website to determine the tagging SNPs, specifically the HapMap Genome Browser release#16 (Phase 1—full data set). Within the program, we configured the Annotate tag SNP Picker as follows: the R2 cutoff for tagging SNP selection was .8 and the minor allele frequency cutoff was 0.2. Genotypes were classified as either variant negative or variant positive. Variant positive genotypes were either homozygous variant or heterozygous. Variant negative genotypes were homozygous wild type.
Angiographic Vasospasm Demographic Information We extracted demographic data from the medical record, including age, race, sex, type of intervention (surgical clipping vs. endovascular aneurysm coiling), Fisher grade, and Hunt & Hess grade. Both Fisher grade and Hunt & Hess grade were assigned by the admitting neurosurgeon.
Data Collection CSF was collected twice daily for up to 14 days, while the ventriculostomy or lumbar drain was necessary for clinical care. Based on our recent report that the ET-1 exposure rate 72 hr before angiography was a significant predictor of angiographic vasospasm (Thampatty et al., 2011), only the CSF samples collected in the 72 hr before angiography were used for the present study. All CSF samples were stored at 80 C until the time of ET-1 analysis. ET-1 levels were quantified using QuantiGlo Human Endothelin-1 Immunoassay kit (R & D Systems, Minneapolis, MN) with a modified standard curve as described in Thampatty et al. (2011). The ET-1 exposure rate at 72 hr prior to angiography was calculated to include the total amount of ET-1 eliminated per unit of time to account for differences in CSF drainage using the following formula: Exposure rate (pg/hr) ¼
ET-1 acts directly on the cerebral blood vessels to induce vasoconstriction. Our previous work confirmed our belief that prolonged exposure to ET-1 in the CSF promotes angiographic vasospasm (Thampatty et al., 2011). In the present study, angiographic vasospasm was determined from cerebral angiograms read and coded by neurosurgeons. Angiograms were performed as standard of care on admission, as postoperative follow-up care, and/or in the event of neurological deterioration. The angiograms used for this project were performed an average of 151.4 + 53.6 hr after hemorrhage. In the angiographic vasospasm positive group (n ¼ 11), the average time from hemorrhage to angiogram was 161.2 + 61.5 hr. In the angiographic vasospasm negative group (n ¼ 21), the average time from hemorrhage to angiogram was 146.3 + 49.7 hr. These findings were dichotomized as vasospasm negative (0–24% narrowing of cerebral blood vessels) or vasospasm positive ( 25% narrowing of cerebral blood vessels).
Statistical Analyses IBM SPSS version 20.0 was used for all analyses. Demographics were described using descriptive statistics. To evaluate the relationship between ET-1 levels and ET-1 gene polymorphisms, independent samples Mann–Whitney U test (nonparametric chi-square test) was used. Since this was an
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Table 1. Genotype Distribution for the Endothelin-1 gene (N ¼ 32). Genotype Frequencies Variant Negative SNP
Variant Positive
Homozygous Wild-Type
rs5370 rs2071943 rs1626492 rs5369 rs1476046 rs2070699 rs6912834 rs3087459 rs1800541
21 21 15 23 19 8 27 20 21
Heterozygous
(G/G) (G/G) (G/G) (G/G) (G/G) (G/G) (A/A) (A/A) (T/T)
9 10 16 9 12 17 5 11 10
Homozygous Variant
HW p value
2 (T/T) 1 (A/A) 1 (A/A) 0 (A/A) 1 (A/A) 7 (T/T) 0 (G/G) 1 (C/C) 1 (G/G)
.458 .885 .181 .355 .583 .719 .632 .727 .885
(G/T) (A/G) (A/G) (A/G) (A/G) (G/T) (G/T) (A/C) (G/T)
Note. HW ¼ Hardy Weinberg; SNP ¼ single-nucleotide polymorphism.
Table 2. Endothelin (ET)-1 Exposure Rate by ET-1 Genotype. 72 hr Before Angiography SNP and Genotype rs2070699 Variant negative Variant positive rs5370 Variant negative Variant positive
N
Mean (SD), pg/hr
8 24
11.36 (7.35) 4.02 (2.66)
21 11
4.16 (2.65) 9.10 (7.40)
p
48 hr Before Angiography Mean (SD), pg/hr
.004
p
24 hr Before Angiography Mean (SD), pg/hr
.004 9.40 (6.77) 3.40 (2.40)
.051
.002 8.77 (5.49) 3.47 (2.70)
.051 3.44 (2.32) 7.67 (6.58)
p
.039 3.43 (2.40) 7.79 (5.72)
Note. SD ¼ standard deviation; SNP ¼ single nucleotide polymorphism.
exploratory study, a significant difference was defined as p .05.
Results Participants were 32 non-Hispanic White patients, predominantly female (n ¼ 20; 62.5%). The mean age of the subject was 56.34 + 10.64 years. The genotype distributions are presented in Table 1. All SNPs were in Hardy–Weinberg equilibrium. ET-1 exposure rates were evaluated in CSF for the 72 hr before angiographic vasospasm was measured. In most cases the angiography was performed due to neurological deterioration. There was a significant difference in CSF ET-1 exposure rates at 72 hr before angiography between the variant positive and the variant negative groups of the rs2070699 SNP and a trend toward significance between the variant positive and the variant negative groups of the rs5370 SNP (Table 2). The remaining seven SNPs measured (rs2071943, rs1626492, rs5369, rs1476046, rs6912834, rs3087459, rs1800541) were not significantly associated with ET-1 exposure rate (data not shown). The variant negative group of SNP rs2070699 was associated with a significantly higher ET-1 exposure rate at 72 hr prior to measurement of angiographic vasospasm when compared to the variant positive group (Figure 1). This group
Figure 1. Rate of exposure to endothelin (ET)-1 of patients with the rs2070699 single-nucleotide polymorphism (SNP) of the ET-1 gene 72 hr before angiography. Variant negative group n ¼ 8; variant positive group n ¼ 24.
was also associated with significantly higher ET-1 levels at 24 and 48 hr before angiographic vasospasm was measured. An inverse relationship was seen with the variant negative
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Figure 2. Rate of exposure to endothelin (ET)-1 of patients with the rs5370 single-nucleotide polymorphism (SNP) of the ET-1 gene 72 hr before angiography. Variant negative group n ¼ 21; variant positive group n ¼ 11.
group of SNP rs5370, which was associated with a trend toward a significantly lower ET-1 exposure rate when compared to the variant positive group (Figure 2). Once again this trend was also seen at 48 hr before angiographic vasospasm was measured. At 24 hr before angiographic vasospasm was measured, a significantly lower ET-1 exposure rate was associated with the variant negative group when compared to the variant positive group.
Discussion This study is the first to report differences in ET-1 CSF exposure rates related to polymorphisms of the ET-1 gene in the aSAH population. There was one ET-1 SNP associated with differences in ET-1 CSF exposure rate at 72 hr before angiography (rs2070699) and one SNP with a trend toward significance. ET-1 is one of the most important vasoconstrictors in the human body and has been implicated as a mediator of cerebral vasospasm following aSAH (Kessler et al., 2005; Mascia et al., 2001; Seifert, Loffler, Zimmermann, Roux, & Stolke, 1995; Suzuki et al., 1990; Yamaji et al., 1990). These differences in ET-1 genotype associated with ET-1 exposure rate 72 hr before angiography may influence the risk of angiographic vasospasm. As previously shown, the 72 hr before angiography is an important exposure time point when significant differences in ET-1 levels are detected between those who develop angiographic vasospasm and those who do not (Thampatty et al., 2011). The significant differences/trends we detected at 24 and 48 hr before angiography in the present study (Table 2) strengthen our hypothesis that there are differences in ET-1 CSF exposure rates related to polymorphisms of the ET-1 gene. The SNP rs2070699 is a tagging SNP and does not account for a functional change in the mature protein, itself; however,
there have been other studies that have found associations between this SNP and other disease states. Rankinen et al. (2007) found that the minor allele of the rs2070699 SNP had a significant interaction with cardiorespiratory fitness and a lower risk of hypertension. Specifically, the minor allele of the rs2070699 locus was associated with a lower hypertension risk in those with a low cardiorespiratory fitness level. In a study investigating susceptibility to high-altitude pulmonary edema, Charu et al. (2006) found that the minor allele of the rs2070699 SNP was overrepresented in subjects experiencing this type of pulmonary edema. Finally, MacClellan et al. (2009) found that the rs2070699 SNP was associated with migraine with visual aura among White women. Our finding in the present study that the variant positive allele of this SNP is associated with significantly lower ET-1 CSF exposure rate is novel in the aSAH population but fits well with the previous literature, suggesting that the variant allele may be associated with lower risk of vasoconstriction. The rs5370 SNP is a G-to-T transversion at Position 5665 affecting the 61st nucleotide of Exon 5, corresponding with a Lys/Asn change at Codon 198. This SNP has been associated with other diseases that are influenced by vascular reactivity, such as idiopathic pulmonary arterial hypertension (Vadapalli, Rani, Sastry, & Nallari, 2010), coronary artery disease/highdensity lipoprotein levels (Pare et al., 2007), and hypertension (Jin et al., 2003). These results are consistent with our finding in the present study that the variant positive genotype was associated with a trend toward higher ET-1 levels in the CSF, which would correspond with increased vasoconstriction. The SNP rs5370 has also been associated with vascular reactivity in vivo in a coronary artery bypass population. In a study of pregnant women, Iglarz et al. (2002) found that subjects with the rs5370 homozygous variant (variant positive) had significantly higher plasma ET-1 levels than subjects with the heterozygous or homozygous wild type (variant negative). In addition, this variant allele was associated with raised systolic blood pressure in the same sample (Barden et al., 2001). By contrast, in a study by Tanaka and associates (2004), there were no differences in ET-1 levels either in vitro or in plasma levels associated with the rs5370 SNP in a population of adults with essential hypertension. The differences in the results of these studies may be related to the populations studied. The small sample size of our population in the present study may also have biased our findings such that we were not able to identify a true relationship. Although the findings of the present study are novel because of the focus on ET-1 levels in CSF, there have been two prior studies that found differences in ET-1 levels in relationship with additional polymorphisms that we did not explore. Tanaka et al. (2004) explored the above-mentioned rs5370 SNP (aka Lys198Asn) and the 134delA polymorphism in subjects with essential hypertension. They found no differences in plasma levels of ET-1 between genotypes of the rs5370 SNP. The 134delA polymorphism is a deletion of an adenine in the 5’untranslated region 134 bp downstream of the transcription site. Tanaka et al. found a significant increase in plasma levels of ET-1 in the homozygous/variant negative (3A/3A) group
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compared to the heterozygous/variant positive (3A/4A) group. Popowski and associates (2003) investigated the relationship between ET-1 levels and the adenine insertion variant in the 5’-untranslated region 138 bp downstream of the transcription start site of the ET-1 gene. In this ex vivo study using human umbilical vein endothelial cells, higher levels of ET-1 were associated with the homozygous insertion variant group (variant positive) when compared to the heterozygous (variant positive) and homozygous wild-type groups (variant negative). Although we did not directly investigate these polymorphisms in the present study, it is conceivable that we captured these differences in genotypes by investigating eight tagging SNPs of the ET-1 gene. In theory, these tagging SNPs should account for all of the variation in the ET-1 gene. The findings in the above-mentioned studies may be replicated by using these tagging SNPs that are in linkage disequilibrium with the adenine deletion and insertion polymorphisms mentioned earlier. Some limitations of this study are worth mentioning. We used nonparametric testing due to unequal variance and significantly different sample sizes in the genotype groups. Larger studies need to be conducted to replicate the findings of this study and to possibly use parametric testing. If parametric t-tests were used in subsequent studies using a power of .80 and a two-tailed a of .05, a sample size of 52 would be needed to detect large effect sizes (d ¼ .80), 128 to detect medium effect sizes (d ¼ .50), and 786 to detect small effect sizes (d ¼ .20; Cohen, 1988). Another limitation is that these results cannot be generalized to the aSAH population as a whole, but rather are limited to those subjects who had an aSAH, a ventriculostomy, or lumbar drain to provide access to CSF, and an angiography. Finally, the limited sample size prevented the use of multiple linear regression to determine the effects of potential covariates (Fisher grade, gender, age) on the observed relationship. These results provide evidence that genetic polymorphisms of the ET-1 gene are associated with different ET-1 exposure rates in the aSAH population. In addition, this study establishes preliminary evidence of differential directions of effect for the rs2070699 and rs5370 gene variants. As prior research has shown that acute (72 hr before angiogram) elevations in CSF ET-1 levels are associated with angiographic vasospasm (Thampatty et al., 2011), the present results support the value of undertaking larger studies to determine more conclusively whether ET-1 genotype is a predictor of ET-1 levels and risk of vasospasm. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by funding from the National Institutes of Health (NIH, RO1NR004339), the Neuroscience Nursing Foundation, the International Society of Nurses in Genetics, and Sigma Theta Tau.
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Endothelin-1 Gene Polymorphisms Influence Cerebrospinal Fluid Endothelin-1 Levels Following Aneurysmal Subarachnoid Hemorrhage
Biological Research for Nursing 2015, Vol. 17(2) 185-190 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1099800414536261 brn.sagepub.com
Matthew J. Gallek, PhD, RN1, Sheila A. Alexander, PhD, RN2, Elizabeth Crago, PhD, RN2, Paula R. Sherwood, PhD, RN2, Megan Klamerus, MS3, Michael B. Horowitz, MD4, Samuel M. Poloyac, PharmD, PhD5, and Yvette Conley, PhD2
Abstract Aneurysmal subarachnoid hemorrhage is a type of stroke with high morbidity and mortality. Increased endothelin-1 (ET-1) levels have been associated with increased risk of cerebral vasospasm, which is associated with increased morbidity. The purpose of this study was to investigate the relationships between ET-1 genotypes and ET-1 protein levels in cerebrospinal fluid (CSF) measured 72 hr before angiographic vasospasm measurement in subjects at high risk of cerebral vasospasm. Specifically, this study evaluated the differences between variant positive and variant negative groups of nine different ET-1 single-nucleotide polymorphisms (SNPs) in relationship with the ET-1 protein exposure rate. The CSF ET-1 protein levels were quantified using enzyme-linked immunosorbent assay. One functional SNP and eight ET-1 tagging SNPs were selected because they represent genetic variability in the entire ET-1 gene. The variant negative group of SNP rs2070699 was associated with a significantly higher ET-1 exposure rate than the variant positive group (p ¼ 0.004), while the variant positive group of the rs5370 group showed a trend toward association with a higher ET-1 exposure rate (p ¼ 0.051). Other SNPs were not informative. This is the first study to show differences in ET-1 exposure rate 72 hr before angiography in relation to ET-1 genotypes. These exploratory findings need to be replicated in a larger study; if replicated, these differences in genotypes may be a way to inform clinicians of those patients at a higher risk of increased ET-1 protein levels, which may lead to a higher risk of angiographic vasospasm. Keywords subarachnoid hemorrhage, endothelin-1, genetics
Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating type of stroke that occurs most often when a cerebral aneurysm ruptures. It affects nearly 30,000 people a year and has a mortality rate of approximately 30% (Nieuwkamp et al., 2009). A major cause of morbidity in this population is delayed cerebral ischemia related to cerebral vasospasm. Angiographic vasospasm occurs in up to 70% of aSAH patients (Dorsch, 2002). Endothelin (ET), a potent vasoconstrictor, is hypothesized to contribute to cerebral vasospasm. Research has shown an association between increased cerebrospinal fluid (CSF) ET-1 levels and angiographic vasospasm (Kessler, Pacheco, Lozzi, de Araujo, & Onishi, 2005; Mascia et al., 2001; Thampatty et al., 2011). Further research has shown that polymorphisms of the ET-1 gene influence levels of the ET-1 protein, but these studies have only included hypertensive patients without aSAH and measured ET-1 only in plasma (Barden et al., 2001; Tanaka,
Kamide, Takiuchi, Kawano, & Miyata, 2004). The purpose of the present study was to investigate the relationships between ET-1 protein levels in CSF measured 72 hr before angiographic vasospasm measurement and ET-1 genotypes in our sample of aSAH patients.
1
College of Nursing, University of Arizona, Tucson, AZ, USA School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA 3 Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA 4 Pennsylvania Brain and Spine Institute, Allegheny Health Network, Pittsburgh, PA, USA 5 School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA 2
Corresponding Author: Matthew J. Gallek, PhD, RN, The University of Arizona, College of Nursing, 1305 N Martin Ave, PO Box 210203, Tucson, AZ 85721, USA. Email: [email protected]
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Biological Research for Nursing 17(2) Concentration of ET-1 protein in the sample (pg/ml) CSF drainage (ml)/Time period (hr)
Material and Method Participants Participants were a subset of individuals who were recruited as part of an ongoing National Institutes of Health–funded study (R01NR004339) following approval by the University of Pittsburgh Institutional Review Board and after obtaining informed consent from family members or, in rare cases, participants themselves. Participants were recruited by hospital staff referral from the neurovascular intensive care unit (ICU) at the University of Pittsburgh Medical Center from September 2000 to April 2006. Inclusion criteria for the parent study were (1) adult patients with the diagnosis of SAH with an aneurysm verified by computerized tomographic angiography or digital subtraction angiography and (2) a Fisher grade > 1. For the present study participants must also have had a ventriculostomy or lumbar drain for CSF collection, angiography for cerebral vasospasm detection, and be non-Hispanic Whites. Although patients of all ethnic backgrounds were included in the recruiting process for the parent study, we limited analyses of our data for the present study to non-Hispanic Whites to address population stratification, given that representation of other races and ethnicities were not large enough to permit separate analyses for those groups. Exclusion criteria included (1) preexisting neurological disorder, (2) traumatic SAH, (3) mycotic aneurysm, or (4) arteriovenous malformation. All participants received standard medical care in the ICU, including triple-H therapy (hypertension, hypervolemia, and hemodilution), nimodipine, and early intervention to repair the aneurysm.
Genotyping Whole blood (3 cc) was collected from the participants within 48 hr of admission for DNA extraction using a simple salting out procedure (Miller, Dykes, & Polesky, 1988). Genotyping of the ET-1 single-nucleotide polymorphisms (SNPs) was completed using the ABI Prism1 7000 Sequence Detection System (Applied Biosystems, USA) and TaqMan1 allele discrimination assays (Applied Biosystems, USA). We chose the rs5370 SNP because it is a functional SNP that has been associated with other diseases. It is a G-to-T transversion at Position 5665 affecting the 61st nucleotide of Exon 5, corresponding with a Lys/Asn change at Codon 198. The remaining eight SNPs we genotyped were tagging SNPs and as such represent known variability within the entire gene. We consulted the HapMap.org website to determine the tagging SNPs, specifically the HapMap Genome Browser release#16 (Phase 1—full data set). Within the program, we configured the Annotate tag SNP Picker as follows: the R2 cutoff for tagging SNP selection was .8 and the minor allele frequency cutoff was 0.2. Genotypes were classified as either variant negative or variant positive. Variant positive genotypes were either homozygous variant or heterozygous. Variant negative genotypes were homozygous wild type.
Angiographic Vasospasm Demographic Information We extracted demographic data from the medical record, including age, race, sex, type of intervention (surgical clipping vs. endovascular aneurysm coiling), Fisher grade, and Hunt & Hess grade. Both Fisher grade and Hunt & Hess grade were assigned by the admitting neurosurgeon.
Data Collection CSF was collected twice daily for up to 14 days, while the ventriculostomy or lumbar drain was necessary for clinical care. Based on our recent report that the ET-1 exposure rate 72 hr before angiography was a significant predictor of angiographic vasospasm (Thampatty et al., 2011), only the CSF samples collected in the 72 hr before angiography were used for the present study. All CSF samples were stored at 80 C until the time of ET-1 analysis. ET-1 levels were quantified using QuantiGlo Human Endothelin-1 Immunoassay kit (R & D Systems, Minneapolis, MN) with a modified standard curve as described in Thampatty et al. (2011). The ET-1 exposure rate at 72 hr prior to angiography was calculated to include the total amount of ET-1 eliminated per unit of time to account for differences in CSF drainage using the following formula: Exposure rate (pg/hr) ¼
ET-1 acts directly on the cerebral blood vessels to induce vasoconstriction. Our previous work confirmed our belief that prolonged exposure to ET-1 in the CSF promotes angiographic vasospasm (Thampatty et al., 2011). In the present study, angiographic vasospasm was determined from cerebral angiograms read and coded by neurosurgeons. Angiograms were performed as standard of care on admission, as postoperative follow-up care, and/or in the event of neurological deterioration. The angiograms used for this project were performed an average of 151.4 + 53.6 hr after hemorrhage. In the angiographic vasospasm positive group (n ¼ 11), the average time from hemorrhage to angiogram was 161.2 + 61.5 hr. In the angiographic vasospasm negative group (n ¼ 21), the average time from hemorrhage to angiogram was 146.3 + 49.7 hr. These findings were dichotomized as vasospasm negative (0–24% narrowing of cerebral blood vessels) or vasospasm positive ( 25% narrowing of cerebral blood vessels).
Statistical Analyses IBM SPSS version 20.0 was used for all analyses. Demographics were described using descriptive statistics. To evaluate the relationship between ET-1 levels and ET-1 gene polymorphisms, independent samples Mann–Whitney U test (nonparametric chi-square test) was used. Since this was an
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Table 1. Genotype Distribution for the Endothelin-1 gene (N ¼ 32). Genotype Frequencies Variant Negative SNP
Variant Positive
Homozygous Wild-Type
rs5370 rs2071943 rs1626492 rs5369 rs1476046 rs2070699 rs6912834 rs3087459 rs1800541
21 21 15 23 19 8 27 20 21
Heterozygous
(G/G) (G/G) (G/G) (G/G) (G/G) (G/G) (A/A) (A/A) (T/T)
9 10 16 9 12 17 5 11 10
Homozygous Variant
HW p value
2 (T/T) 1 (A/A) 1 (A/A) 0 (A/A) 1 (A/A) 7 (T/T) 0 (G/G) 1 (C/C) 1 (G/G)
.458 .885 .181 .355 .583 .719 .632 .727 .885
(G/T) (A/G) (A/G) (A/G) (A/G) (G/T) (G/T) (A/C) (G/T)
Note. HW ¼ Hardy Weinberg; SNP ¼ single-nucleotide polymorphism.
Table 2. Endothelin (ET)-1 Exposure Rate by ET-1 Genotype. 72 hr Before Angiography SNP and Genotype rs2070699 Variant negative Variant positive rs5370 Variant negative Variant positive
N
Mean (SD), pg/hr
8 24
11.36 (7.35) 4.02 (2.66)
21 11
4.16 (2.65) 9.10 (7.40)
p
48 hr Before Angiography Mean (SD), pg/hr
.004
p
24 hr Before Angiography Mean (SD), pg/hr
.004 9.40 (6.77) 3.40 (2.40)
.051
.002 8.77 (5.49) 3.47 (2.70)
.051 3.44 (2.32) 7.67 (6.58)
p
.039 3.43 (2.40) 7.79 (5.72)
Note. SD ¼ standard deviation; SNP ¼ single nucleotide polymorphism.
exploratory study, a significant difference was defined as p .05.
Results Participants were 32 non-Hispanic White patients, predominantly female (n ¼ 20; 62.5%). The mean age of the subject was 56.34 + 10.64 years. The genotype distributions are presented in Table 1. All SNPs were in Hardy–Weinberg equilibrium. ET-1 exposure rates were evaluated in CSF for the 72 hr before angiographic vasospasm was measured. In most cases the angiography was performed due to neurological deterioration. There was a significant difference in CSF ET-1 exposure rates at 72 hr before angiography between the variant positive and the variant negative groups of the rs2070699 SNP and a trend toward significance between the variant positive and the variant negative groups of the rs5370 SNP (Table 2). The remaining seven SNPs measured (rs2071943, rs1626492, rs5369, rs1476046, rs6912834, rs3087459, rs1800541) were not significantly associated with ET-1 exposure rate (data not shown). The variant negative group of SNP rs2070699 was associated with a significantly higher ET-1 exposure rate at 72 hr prior to measurement of angiographic vasospasm when compared to the variant positive group (Figure 1). This group
Figure 1. Rate of exposure to endothelin (ET)-1 of patients with the rs2070699 single-nucleotide polymorphism (SNP) of the ET-1 gene 72 hr before angiography. Variant negative group n ¼ 8; variant positive group n ¼ 24.
was also associated with significantly higher ET-1 levels at 24 and 48 hr before angiographic vasospasm was measured. An inverse relationship was seen with the variant negative
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Figure 2. Rate of exposure to endothelin (ET)-1 of patients with the rs5370 single-nucleotide polymorphism (SNP) of the ET-1 gene 72 hr before angiography. Variant negative group n ¼ 21; variant positive group n ¼ 11.
group of SNP rs5370, which was associated with a trend toward a significantly lower ET-1 exposure rate when compared to the variant positive group (Figure 2). Once again this trend was also seen at 48 hr before angiographic vasospasm was measured. At 24 hr before angiographic vasospasm was measured, a significantly lower ET-1 exposure rate was associated with the variant negative group when compared to the variant positive group.
Discussion This study is the first to report differences in ET-1 CSF exposure rates related to polymorphisms of the ET-1 gene in the aSAH population. There was one ET-1 SNP associated with differences in ET-1 CSF exposure rate at 72 hr before angiography (rs2070699) and one SNP with a trend toward significance. ET-1 is one of the most important vasoconstrictors in the human body and has been implicated as a mediator of cerebral vasospasm following aSAH (Kessler et al., 2005; Mascia et al., 2001; Seifert, Loffler, Zimmermann, Roux, & Stolke, 1995; Suzuki et al., 1990; Yamaji et al., 1990). These differences in ET-1 genotype associated with ET-1 exposure rate 72 hr before angiography may influence the risk of angiographic vasospasm. As previously shown, the 72 hr before angiography is an important exposure time point when significant differences in ET-1 levels are detected between those who develop angiographic vasospasm and those who do not (Thampatty et al., 2011). The significant differences/trends we detected at 24 and 48 hr before angiography in the present study (Table 2) strengthen our hypothesis that there are differences in ET-1 CSF exposure rates related to polymorphisms of the ET-1 gene. The SNP rs2070699 is a tagging SNP and does not account for a functional change in the mature protein, itself; however,
there have been other studies that have found associations between this SNP and other disease states. Rankinen et al. (2007) found that the minor allele of the rs2070699 SNP had a significant interaction with cardiorespiratory fitness and a lower risk of hypertension. Specifically, the minor allele of the rs2070699 locus was associated with a lower hypertension risk in those with a low cardiorespiratory fitness level. In a study investigating susceptibility to high-altitude pulmonary edema, Charu et al. (2006) found that the minor allele of the rs2070699 SNP was overrepresented in subjects experiencing this type of pulmonary edema. Finally, MacClellan et al. (2009) found that the rs2070699 SNP was associated with migraine with visual aura among White women. Our finding in the present study that the variant positive allele of this SNP is associated with significantly lower ET-1 CSF exposure rate is novel in the aSAH population but fits well with the previous literature, suggesting that the variant allele may be associated with lower risk of vasoconstriction. The rs5370 SNP is a G-to-T transversion at Position 5665 affecting the 61st nucleotide of Exon 5, corresponding with a Lys/Asn change at Codon 198. This SNP has been associated with other diseases that are influenced by vascular reactivity, such as idiopathic pulmonary arterial hypertension (Vadapalli, Rani, Sastry, & Nallari, 2010), coronary artery disease/highdensity lipoprotein levels (Pare et al., 2007), and hypertension (Jin et al., 2003). These results are consistent with our finding in the present study that the variant positive genotype was associated with a trend toward higher ET-1 levels in the CSF, which would correspond with increased vasoconstriction. The SNP rs5370 has also been associated with vascular reactivity in vivo in a coronary artery bypass population. In a study of pregnant women, Iglarz et al. (2002) found that subjects with the rs5370 homozygous variant (variant positive) had significantly higher plasma ET-1 levels than subjects with the heterozygous or homozygous wild type (variant negative). In addition, this variant allele was associated with raised systolic blood pressure in the same sample (Barden et al., 2001). By contrast, in a study by Tanaka and associates (2004), there were no differences in ET-1 levels either in vitro or in plasma levels associated with the rs5370 SNP in a population of adults with essential hypertension. The differences in the results of these studies may be related to the populations studied. The small sample size of our population in the present study may also have biased our findings such that we were not able to identify a true relationship. Although the findings of the present study are novel because of the focus on ET-1 levels in CSF, there have been two prior studies that found differences in ET-1 levels in relationship with additional polymorphisms that we did not explore. Tanaka et al. (2004) explored the above-mentioned rs5370 SNP (aka Lys198Asn) and the 134delA polymorphism in subjects with essential hypertension. They found no differences in plasma levels of ET-1 between genotypes of the rs5370 SNP. The 134delA polymorphism is a deletion of an adenine in the 5’untranslated region 134 bp downstream of the transcription site. Tanaka et al. found a significant increase in plasma levels of ET-1 in the homozygous/variant negative (3A/3A) group
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compared to the heterozygous/variant positive (3A/4A) group. Popowski and associates (2003) investigated the relationship between ET-1 levels and the adenine insertion variant in the 5’-untranslated region 138 bp downstream of the transcription start site of the ET-1 gene. In this ex vivo study using human umbilical vein endothelial cells, higher levels of ET-1 were associated with the homozygous insertion variant group (variant positive) when compared to the heterozygous (variant positive) and homozygous wild-type groups (variant negative). Although we did not directly investigate these polymorphisms in the present study, it is conceivable that we captured these differences in genotypes by investigating eight tagging SNPs of the ET-1 gene. In theory, these tagging SNPs should account for all of the variation in the ET-1 gene. The findings in the above-mentioned studies may be replicated by using these tagging SNPs that are in linkage disequilibrium with the adenine deletion and insertion polymorphisms mentioned earlier. Some limitations of this study are worth mentioning. We used nonparametric testing due to unequal variance and significantly different sample sizes in the genotype groups. Larger studies need to be conducted to replicate the findings of this study and to possibly use parametric testing. If parametric t-tests were used in subsequent studies using a power of .80 and a two-tailed a of .05, a sample size of 52 would be needed to detect large effect sizes (d ¼ .80), 128 to detect medium effect sizes (d ¼ .50), and 786 to detect small effect sizes (d ¼ .20; Cohen, 1988). Another limitation is that these results cannot be generalized to the aSAH population as a whole, but rather are limited to those subjects who had an aSAH, a ventriculostomy, or lumbar drain to provide access to CSF, and an angiography. Finally, the limited sample size prevented the use of multiple linear regression to determine the effects of potential covariates (Fisher grade, gender, age) on the observed relationship. These results provide evidence that genetic polymorphisms of the ET-1 gene are associated with different ET-1 exposure rates in the aSAH population. In addition, this study establishes preliminary evidence of differential directions of effect for the rs2070699 and rs5370 gene variants. As prior research has shown that acute (72 hr before angiogram) elevations in CSF ET-1 levels are associated with angiographic vasospasm (Thampatty et al., 2011), the present results support the value of undertaking larger studies to determine more conclusively whether ET-1 genotype is a predictor of ET-1 levels and risk of vasospasm. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by funding from the National Institutes of Health (NIH, RO1NR004339), the Neuroscience Nursing Foundation, the International Society of Nurses in Genetics, and Sigma Theta Tau.
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