Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Biol Res Nurs. 2012 Sep 19;15(4):390–397. doi: 10.1177/1099800412459674

Endothelin-1 and Endothelin Receptor Gene Variants and Their Association With Negative Outcomes Following Aneurysmal Subarachnoid Hemorrhage

Matthew Gallek 1, Sheila Alexander 2, Elizabeth Crago 2, Paula Sherwood 2, Michael Horowitz 2, Samuel Poloyac 2, Yvette Conley 2
PMCID: PMC3758781  NIHMSID: NIHMS475314  PMID: 22997346

Abstract

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease that affects approximately 30,000 people a year in the United States. Delayed cerebral ischemia (DCI) and cerebral vasospasm (CV) are common complications after aSAH. In addition, aSAH patients have a high risk of poor long-term outcomes. Endothelin-1 (ET-1), a potent vasoconstrictor, or its two types of receptors, ET receptor A (ETA ) and ET receptor B (ETB), may play a role in the pathogenesis of DCI and CV. Genetic variations within the ET-1, ETA, or ETB genes may also account for variance observed in the outcomes of aSAH patients. The purpose of this study was to describe the distribution of the Lys198Asn polymorphism, a known functional SNP in the ET-1 gene, and tagging SNPs of the ET-1, ETA, and ETB genes in individuals recovering from aSAH. This study also investigated the relationships among the ET polymorphisms, DCI, and global functional outcomes measured at 3 and 6 months after aSAH. Participants included individuals aged 18–75 years with a diagnosis of aSAH. There was a trend found between the variant allele of an ET-1 SNP (rs6912834) and angiographic vasospasm. There were also associations found between two ETB SNPs (rs9574124 and rs3027111) and poor outcomes as measured by the Glasgow Outcome scale at 3 months. These findings support the role of ET-1 and ETB in recovery following aSAH.

Keywords: aneurysmal subarachnoid hemorrhage, cerebral vasospasm, delayed cerebral ischemia, endothelin-1, endothelin receptor A, endothelin receptor B, functional outcomes

There are approximately 25,000 cases of subarachnoid hemorrhage (SAH) in the United States each year (Roger et al., 2011), most of which occur in women at an average age of 50 years (Anderson, Anderson, & Bonita, 2000). Ruptured cerebral aneurysms are the cause for 85% of spontaneous SAH cases (van Gijn, Kerr, & Rinkel, 2007). The morbidity and mortality rates of aneurysmal subarachnoid hemorrhage (aSAH) have decreased little over the past 40 years (Stegmayr, Eriksson, & Asplund, 2004) despite the development of new early intervention methods designed to improve survival rates following the initial injury. While early intervention has decreased the risk of aneurysmal rebleed, the frequency of secondary injuries has become evident, and the survival rate from aSAH has not improved.

Cerebral vasospasm (CV), a decrease in diameter of the lumen of cerebral blood vessels, is one mechanism that can lead to delayed cerebral ischemia (DCI) following aSAH, furthering neurological damage and possibly causing ischemic stroke. Worse clinical outcomes have been associated with the presence of CV compared to aSAH patients without CV (Goto & Yamagata, 2006; Ratsep & Asser, 2001). CV has been correlated with a 1.5- to 3-fold increase in mortality in the first 2 weeks after aSAH (Treggiari-Venzi et al., 2001). Angio-graphic vasospasm or arterial vasospasm indicating vascular constriction is present in approximately 70% of aSAH patients (Dorsch, 2002). Approximately 20–30% of these patients display signs of neurological deficit termed symptomatic or clinical vasospasm.

While DCI may be caused by factors such as CV or arterial narrowing, it can also occur in the absence of radiologically confirmed arterial narrowing (Vergouwen et al., 2010). Researchers have investigated numerous possible causal factors for DCI (Cohen & Vanhoutte, 1995; Hatake, Wakabayashi, Kakishita, & Hishida, 1992; Kasuya et al., 1995; Kim, Schini, Sundt, & Vanhoutte, 1992; Onoue et al., 1995; Sobey, Heistad, & Faraci, 1996; Yamamoto, Nishizawa, Yokoyama, Ryu, & Uemura, 1997). Endothelin-1 (ET-1) is a long-lasting vasoconstrictor produced by the endothelial cells of the vasculature (Petzold, Einhaupl, Dirnagl, & Dreier, 2003; Suzuki, Sato, Suzuki, Oka, et al., 1990). Suzuki and associates reported increased levels of ET-1 in the cerebrospinal fluid (CSF) of aSAH patients (Suzuki, Sato, Suzuki, Takekoshi, et al., 1990). Other researchers have found that CSF ET-1 levels are associated with the development of CV after aSAH (Kessler, Pacheco, Lozzi, de Araujo, & Onishi, 2005; Thampatty et al., 2011), thus supporting the role of ET-1 in the pathogenesis of CV and DCI.

Researchers have investigated a number of predictors of DCI, including amount and distribution of blood on computed tomography (CT) scan (categorized by Fisher grade), age, hypertension, cigarette smoking, and cocaine use (Conway & Tamargo, 2001; Qureshi et al., 2001); however, none of these risk factors accurately predict the occurrence of DCI. Determination of genetic susceptibility may allow for more accurate identification of aSAH patients at high risk of poor outcomes, thus allowing for closer monitoring and more aggressive interventions. The purpose of the present study was to describe the distribution of the Lys198Asn polymorphism, a known functional SNP in the ET-1 gene, and tagging SNPs of the ET-1, ETA, and ETB genes in individuals recovering from aSAH. We also investigated the relationships among the ET polymorphisms, DCI, and global functional outcomes measured at 3 and 6 months after aSAH.

Materials and Methods

Subjects

Following institutional review board approval and after obtaining informed consent from the patient or next of kin, we recruited 250 patients as part of an ongoing National Institutes of Health (NIH)-funded study (RO1NR004339). Inclusion criteria were adult patients (ages 18–75 years) with the diagnosis of aSAH verified by digital subtraction angiography or CT angiography (CTA) with a Fisher grade > 1 and/or a Hunt and Hess score > 1. We did not exclude any people based on ethnic background in the recruiting process. However, because of known allele frequency distribution difference based on ancestry, we limited our analyses for this project to non-Hispanic Whites in an attempt to address population stratification. We excluded patients from the study if they had a preexisting neurological disorder. We collected a blood sample from each patient to obtain a deoxyribonucleic acid (DNA) sample for analysis.

Participants were admitted to the neurovascular intensive care unit (NV-ICU) at the University of Pittsburgh Medical Center (UPMC) from September 2000 to April 2006. All received standard nursing and medical care in the NV-ICU, including triple-H therapy (hypertension, hypervolemia, and hemodilution), nimodipine, and early surgical or endovascular intervention. We used this sample of 250 patients to describe the distribution of ET-1, ETA, and ETB polymorphisms in the aSAH population.

Measures

Demographic information

We collected age in years, race, sex, Fisher grade, and Hunt and Hess grade from medical records. The admitting neurosurgeon assigned the Fisher and Hunt and Hess grades.

DCI

For the purpose of this study, we used the definition of DCI presented in Crago et al. (2011), such that, for DCI to occur there must be a neurological deterioration in the presence of impaired cerebral blood flow. We defined neurologic deterioration as a change in level of consciousness, pupil changes, presence of a new focal neurologic deficit, or worsening Glasgow Coma scale or National Institutes of Health Stroke scale (NIHSS) scores. We did not classify neurological deterioration as DCI if the deterioration could be associated with medication administration (e.g., narcotics), hydrocephalus, or any other non-blood-flow causes of deterioration. We assessed cerebral blood flow by cerebral angiography, transcranial Doppler (TCD) and CT or magnetic resonance imaging perfusion scans.

Angiographic vasospasm

Neurosurgeons blinded to participant identity read participants’ cerebral angiograms and coded them as either “negative” (0–24% narrowing of cerebral blood vessels) or “positive” (>25% narrowing of cerebral blood vessels).

To determine CV, we examined the following variables: (1) angiographic results, when present and (2) daily TCD ultrasound results (mean systolic velocities >200 and/or Lindegaard ratios >3) accompanied by clinical deterioration as recorded by the nurse (a decrease in the NIHSS score of 2 or more from the previous assessment; Sloan et al., 1989; Spilker et al., 1997). We classified patients as either (1) clinical vasospasm positive—elevated vessel velocities on TCD ultrasound and/or angiographic vasospasm with corresponding clinical deterioration or (2) clinical vasospasm negative—the absence of neurologic deterioration or neurologic deterioration with no changes in cerebral vessel diameter.

Global functional outcomes

We collected the global functional outcome scores at 3 months and 6 months after aneurysm rupture. The Glasgow Outcome scale (GOS), a clinical observation scale, categorizes functional outcomes into five levels, with 1 indicating death and 5 indicating good recovery (Jennett, 1976). The GOS has wide acceptance and established validity (Jennett & Bond, 1975; Maas, Braakman, Schouten, Minderhoud, & van Zomeren, 1983). The GOS correlates well with severity of illness. Interrater reliability has been reported from 68% to 95% with κ values from .62 to .79 (Gennarelli et al., 1982) and is within the acceptable range when there is adherence to assessment guidelines, administration of a structured interview, and training of examiners (Wilson, Pettigrew, & Teasdale, 1998). We dichomotized scores on the GOS into good outcome (score of 4 to 5) and poor outcome (score of 1 to 3).

We used the Modified Rankin Scale (MRS) as a measure of functional recovery. This scale incorporates mental and physical adaptations to the neurological deficits following a neurological injury. The MRS is easy to use and has been widely adopted for use in stroke trials. Scores range from 0 (no symptoms at all) to 6 (death). Researchers have reported the interobserver κ to be .56 and weighted κ to be .91 (Sulter, Steen, & De Keyser, 1999). We also dichotomized the MRS scores into good outcome (score of 0 to 3) and poor outcome (score of 4 to 6). A trained neuropsychological technician who was blinded to genotype collected both global functional outcome scores.

Mortality

We obtained mortality status from medical records or caregiver reports at 3 and 6 months.

Procedures

DNA specimens

We drew 3 cc of whole blood from subjects within 48 hr of their admission. The tube of blood we delivered to the genetics laboratory had no personal identifiers and was labeled only with the study participant's identification number, date, and time of sample collection. We logged in and centrifuged the sample, removed the white cells, and extracted DNA from the white cells using a simple salting out procedure (Miller, Dykes, & Polesky, 1988). We stored all DNA in 1X TE buffer at 4 °C until genotyping was performed.

Genotyping

We used an ABI Prism® 7000 Sequence Detection System (Applied Bioscience, Carlsbad, CA, USA) to conduct allele discrimination using TaqMan® allele discrimination assays. We classified tagging SNPs of ET-1, ETA, ETB, and Lys198Asn into variant positive or variant negative. Variant positive genotypes were either homozygous variant or heterozygous. Variant negative genotypes were homozygous wild type.

Haplotype calculation

We performed haplotype calculation using THESIAS 3.1 software (Tregouet & Garelle, 2007). For the ETA and ETB genes, we used all SNPs in the calculation for each gene independently. Because of the number of SNPs for the ET-1 gene, we used a “rolling haplotype.” For each haplo-type calculation, we used three SNPs, starting at the 3’ end and moving to the 5’ end of the gene. For example, the first calculation was SNP 1, SNP 2, and SNP 3. The second calculation was SNP 2, SNP 3, and SNP 4.

Statistical Analysis

Statistical Package for the Social Sciences (SPSS) version 16.0 was used for all analyses. Values are presented in the tables and text as mean ± SD. Differences in demographics based on CV status, DCI, outcome scores, and genotypes were investigated using chi-square or Fisher analysis for categorical variables, and independent t-test for continuous variables.

Binary logistic regression was used to evaluate the relationship between genotype and CV or DCI controlling for age and Fisher grade. Outcomes at 3 months and at 6 months were evaluated using multinomial logistic regression. In this analysis, age, Hunt and Hess grade, and CV status were controlled for in the model. A significant difference was defined as a p ≤ .05.

Results

There were 235 subjects with genotype, demographic, DCI, and functional outcome data available. These subjects were predominantly female (n = 165; 70.2%) and all were non-Hispanic Whites. The mean subject age was 53.37 ± 11.23 years. Table 1 presents demographic information in relationship to DCI. There were no significant differences in gender or age between the two groups. However, we did find significant differences between groups when we compared Hunt and Hess grade and Fisher grade. Table 2 presents the genotype distributions. Of the 19 SNPs we investigated, 18 were in Hardy Weinberg equilibrium, with SNP rs1626492 being the one exception. There were no significant differences in genotype distribution of the ET-1, ETA, or ETB SNPs for this sample when compared to the general population.

Table 1.

Demographic Characteristics of Subjects With Outcomes Measurement Categorized by Delayed Cerebral Ischemia (DCI; N = 235)

Characteristic DCI negative (n = 122) DCI positive (n = 113) p value
Sex, female (n [%]) 86 (70.5%) 79 (69.9%) .924
Age (mean [SD]) 53.2 (10.9) 53.55 (11.6) .811
Hunt and Hess grade (n [%]) .007*
    1 23 (18.9%) 13 (11.5%)
    2 46 (37.7%) 24 (21.2%)
    3 37 (30.4%) 48 (42.5%)
    4 12 (9.8%) 20 (17.7%)
    5 4 (3.2%) 8 (7.1%)
Fisher grade (n [%]) .002*
    1 0 (0%) 0 (0%)
    2 49 (40.2%) 25 (22.1%)
    3 61 (50.0%) 63 (55.8%)
    4 12 (9.8%) 25 (22.1%)
Open in a new tab
*

p values considered significant at p ≤ .05.

Table 2.

Genotype Distribution

Genotype
Gene and SNPs Homozygous wild-type Homozygous variant Heterozygous HW p value
Endothelin-1
    rs5370 145 (G/G) 14 (T/T) 74 (G/T) .257
    rs2071943 144 (G/G) 16 (A/A) 73 (A/G) .111
    rs1626492 114 (G/G) 12 (A/A) 107 (A/G) <.001*
    rs5369 174 (G/G) 5 (A/A) 54 (A/G) .769
    rs1476046 138 (G/G) 15 (A/A) 79 (A/G) .265
    rs2070699 72 (G/G) 45 (T/T) 117 (G/T) .944
    rs6912834 182 (A/A) 2 (G/G) 49 (G/T) .410
    rs3087459 153 (A/A) 5 (C/C) 75 (A/C) .294
    rs1800541 154 (T/T) 5 (G/G) 73 (G/T) .357
Endothelin receptor Type A
    rs6841799 115 (C/C) 14 (G/G) 100 (C/G) .239
    rs5342 90 (A/A) 30 (G/G) 111 (A/G) .695
    rs10305860 112 (G/G) 14 (A/A) 102 (A/G) .106
    rs6537484 85 (C/C) 25 (G/G) 116 (C/G) .081
    rs6812093 115 (A/A) 14 (T/T) 102 (A/T) .195
    rs10008744 126 (A/A) 11 (C/C) 91 (A/C) .345
Endothelin receptor Type B
    rs9574124 114 (C/C) 24 (C/G) 93 (G/G) .678
    rs4885493 129 (C/C) 14 (G/G) 73 (C/G) .457
    rs4885491 171 (G/G) 3 (A/A) 48 (A/G) .767
    rs3027111 154 (C/C) 5 (T/T) 61 (C/T) .599
Open in a new tab

Note. HW = Hardy Weinberg equilibrium.

*

Significant (i.e., not in Hardy Weinberg equilibrium).

DCI

All 235 subjects had DCI data available for analyses. There were no significant associations between DCI and genotype while controlling for age and Fisher grade. We conducted haplotype analyses on each of the three genes investigated (ET-1, ETA, and ETB). There were no haplotypes that were significantly associated with the occurrence of DCI.

Angiographic CV

There were 146 subjects with angiographic data. The subjects were mostly female (n = 105, 71.9%). The mean age of the subjects was 53.62 ± 11.4 years. Of these 146 subjects, 63 (43.2%) had angiographic vasospasm. There was a trend associated with subjects who were variant allele positive for the rs6912834 SNP (ET-1 gene) to develop angiographic vasospasm (p = .051). However, there were no other SNPs associated with angiographic vasospasm. Age and Fisher grade were controlled for in this analysis. There were no haplotypes of the three genes investigated that were significantly associated with the occurrence of DCI.

GOS/MRS

At the 3-month outcome measure, there were 191 subjects with outcome data available. The majority of these subjects were female (134, 70.2%), and their mean age was 53.70 ± 10.9 years. We present the GOS/MRS scores in relation to DCI in Table 3. A total of 33 (17.3%) subjects had a poor outcome as measured by MRS and 37 (19.4%) as measured by GOS. Using binary logistic regression while controlling for age, Hunt and Hess score, and presence of DCI, we found two SNPs with variant alleles associated with a poor score on the GOS: rs9574124 (p = .014) and rs3027111 (p = .015), both of which are on the ET receptor B gene. When using the MRS to score the subjects, we found that only one of the SNP's (rs3027111; p = .038) variant allele was associated with poor outcomes. There were no haplotypes associated with poor outcomes measured by GOS or MRS.

Table 3.

Glasgow Outcome Scale (GOS) and Modified Rankin Scale (MRS) scores and Mortality by Delayed Cerebral Ischemia (DCI; N = 235)

Outcome variable DCI negative n (%) DCI positive n (%) p value
GOS at 3 months .011*
    “Good” outcome 90 (87.4%) 64 (72.7%)
    “Bad” outcome 13 (12.6%) 24 (27.3%)
MRS at 3 months .026*
    “Good” outcome 91 (88.3%) 67 (76.1%)
    “Bad” outcome 12 (11.7%) 21 (23.9%)
Mortality at 3 months .084
    Alive 94 (91.3%) 73 (83.0%)
    Dead 9 (8.7%) 15 (17.0%)
GOS at 6 months .014*
    “Good” outcome 78 (87.6%) 52 (72.2%)
    “Bad” Outcome 11 (12.4%) 20 (27.8%)
MRS at 6 months .014*
    “Good” outcome 78 (87.6%) 52 (72.2%)
    “Bad” outcome 11 (12.4%) 20 (27.8%)
Mortality at 6 months .060*
    Alive 79 (88.8%) 56 (77.8%)
    Dead 10 (11.2%) 16 (22.2%)
Open in a new tab
*

Significant at p < .05.

At 6 months post aneurysm rupture, 161 subjects had GOS and MRS data available. These subjects were predominantly female (114, 70.8%) with a mean age of 54.66 ± 10.9 years. Of these, 31 (19.3%) had poor outcomes measured by both the MRS and the GOS instruments (Table 3). No variant alleles of the SNPs were associated with poor outcomes measured at 6 months. There were also no haplotypes that were associated with poor outcomes at this time period.

Mortality

We collected mortality rates at both 3 and 6 months post aneurysm rupture (Table 3). There were 24 deaths (12.6%) at the 3-month outcome data collection and 26 deaths (16.1%) at the 6-month outcome data collection. The variant allele of SNP rs3027111 (ETB gene) was associated with increased risk of death (p = .018) at 3 months post rupture when controlling for age, Hunt and Hess Score, and DCI. There was a trend toward association for the variant allele of the rs3027111 SNP and death at 6 months (p = .058) when controlling for age, Hunt and Hess Score, and DCI.

Discussion

The major finding of the present study is that there are numerous gene polymorphisms associated with outcomes following aSAH. There was a trend toward association between the minor allele of the rs6912834 SNP (ET-1 gene) and angiographic vasospasm. Poor global functional outcomes measured at 3 months were associated with the minor alleles of the SNPs rs9574124 and rs3027111, both on the ETB gene. Finally, the minor allele of SNP rs3027111 (ETB gene) was associated with a higher risk of mortality at the 3-month outcome measure. All of these findings have clinical relevance. This study is the first to identify minor alleles of the ET-1 and ETB genes that are associated with poor outcomes following aSAH. In the future, these minor alleles may be used as a screening tool to determine which patients may be at higher risk of poor outcomes. Such a tool will allow nurses and other health care professionals to focus resources on these at-risk patients to improve their outcomes following aSAH.

ET-1 is a well-known, potent vasoconstrictor produced by the endothelial cells of the vasculature (Suzuki, Sato, Suzuki, Oka, et al., 1990; Yanagisawa et al., 1988). Researchers have also implicated ET-1 as a mediator of CV and therefore DCI (Mascia et al., 2001; Seifert, Loffler, Zimmermann, Roux, & Stolke, 1995; Yamaji et al., 1990; Yanagisawa et al., 1988). Suzuki and associates reported increased levels of ET-1 in the CSF of aSAH patients (Suzuki, Sato, Suzuki, Takekoshi, et al., 1990). Recent studies showed that increased CSF ET-1 levels are associated with the development of CV after aSAH (Kessler, Pacheco, Lozzi, de Araujo, Onishi, de Mello, et al., 2005), thus supporting the role of ET-1 in the pathogenesis of CV.

Polymorphisms in the ET-1 gene have been investigated in numerous vascular conditions, including essential hypertension (Stevens & Brown, 1995), preeclamptic pregnancy (Barden et al., 2001), cerebral small vessel disease (Gormley, Bevan, Hassan, & Markus, 2005), and arterial stiffness (Iemitsu et al., 2006), to name a few. A functional polymorphism (Lys198Asn) of the gene encoding ET-1 has shown differences in vasculature reactivity (Iglarz et al., 2002). The increase in vasculature reactivity caused by this polymorphism may have an effect on the development or severity of DCI, thereby influencing long-term outcomes in aSAH. Along with this known functional polymorphism, other polymorphisms in the gene for ET-1 may influence DCI and long-term outcomes. It is also important to investigate the genetic variability associated with the receptor, ETA and ETB, polymorphisms as they moderate the effects of ET-1 in the vasculature. The gene that encodes for ETA is located on Chromosome 4. In recent studies, polymorphisms of this receptor have been associated with increased diastolic blood pressure in subjects with essential hypertension (Benjafield, Katyk, & Morris, 2003; Stevens & Brown, 1995). ETA polymorphisms have also been associated with pulse pressure (Nicaud et al., 1999) and prediction of survival in patients with idiopathic dilated cardiomyopathy (Herrmann et al., 2001). The gene for ETB is located on Chromosome 13.

By exploring all of the tagging SNPs of ET-1, ETA, and ETB, it is possible to account for genetic variance in each of the genes, including potentially functional polymorphisms. To date, there are no published studies investigating ET-1, ETA, or ETB tagging SNPs or functional SNPs and their associations with DCI or outcomes following aSAH.

The distribution of the genotypes for the sample of 253 non-Hispanic White subjects in the present study was not significantly different when compared to the HAP-MAP database of the general non-Hispanic White population. From this finding, we conclude that genetic variation in ET-1, ETA, or ETB plays no role in aneurysm formation, development, or rupture. If these genes were involved in aneurysm formation or rupture, we would have expected the genotype distribution to be different in this sample when compared to the general population. There was one SNP that was not in Hardy Weinberg equilibrium. This SNP, rs1626492 (ET-1 gene), may not be in equilibrium for numerous reasons. It may appear to be in genetic drift because of the small number in this sample. Carriers of this SNP may also be at a disadvantage for survival of the original bleed following aneurysm rupture or may be at decreased risk of aneurysm development/rupture.

The genotype distribution of the subset of 191 subjects with 3-month outcome data had a similar distribution to that of the general population. The minor allele of the SNP rs3027111 (ETB gene) was associated with poor outcomes when measured by both the GOS and the MRS. However, the variant allele for SNP rs9574124 (ETB gene) was only associated with a poor outcome when measured by the GOS. This discrepancy is likely due to the difference in sensitivity between the two scales. Subjects with the variant allele for rs3027111 were 3.637 times (95% confidence interval [CI] [1.077, 12.284]) more likely to have a poor outcome when measured by the MRS at 3 months post aneurysm rupture than those without the allele. When outcome was measured by GOS, the subjects were 4.485 times (95% CI [1.346, 14.948]) more likely to have a poor outcome. Subjects with the rs9574124 variant allele were 3.074 times (95% CI = 1.261 to 7.496) more likely to have a poor outcome measured by the GOS at 3 months following aneurysm rupture than those without the allele.

Contrary to our initial hypothesis, our findings show that there were no significant associations between genotype and DCI. DCI is a complicated phenomenon. ET-1 and the ET receptor genes are mainly involved in vasoconstriction. Therefore, it is more likely that these genes would be involved in angiographic CV. Many of our subjects with angiographic CV did not show clinical signs of CV. Therefore, there are other factors (genes/proteins) influencing DCI. Some subjects with angiographic CV are able to compensate through other mechanisms, allowing them to show no signs of DCI, while others do not have the ability to compensate and therefore show signs of DCI.

The genotype distribution of the subset of 146 subjects with angiographic data was also similar to that of the general population. The functional SNP (Lys198Asn) had no association with angiographic CV after aSAH. Therefore, the difference in vascular response to ET-1, as reported by Iglarz et al. (2002), does not appear to play a part in the etiology of CV. After controlling for age and Fisher score, we found a trend for an association between the presence of the variant allele of the tagging SNP rs6912834 (ET-1 gene) and angiographic CV. Subjects with the variant allele were 2.714 times (C.I. = 0.994 to 7.410) more likely to have angiographic vasospasm when compared to subjects without a variant allele. This genotype association supports the role of ET-1 in the development of angiographic CV following aSAH and the proposition that the gene product associated with the variant allele of the rs6912834 SNP may lead to more potent vasoconstriction. While the SNP rs6912834 is within an intron of the ET-1 gene and has no known direct effect on the mature protein, the region of the ET-1 gene that it tags extends to regions of the gene that may affect the mature ET-1 protein. The lack of association in the present study between ETA and ETB with angiographic CV does not rule out the possibility that these genes play a role in CV.

The genotype distribution of the subset of 161 subjects with 6-month outcome data was similar to that of the general population. We found no significant associations between the ET-1, ETA, or ETB genes and functional outcomes measured at 6 months.

Long-term recovery from aSAH is complex and multifactorial. Numerous genes are involved in recovery and cell repair. The ETB receptor gene is one of the genes that are involved in recovery within the first 3 months of aneurysm rupture. However, after that time period, it appears that ETB is not as important in functional recovery. Our findings lead to the conclusion that the tagging SNPs of the ET-1 and ETA genes are most likely not major direct contributors to long-term recovery after aSAH. However, as Thampatty et al. (2011) showed in a previous study, ET-1 protein levels in the CSF are associated with outcomes following aSAH.

Finally, the rs3027111 (ETB gene) variant allele was associated with a higher risk of mortality at the 3-month outcome measure. These subjects were 13.601 times (95% CI [1.550, 119.338]) more likely to die within the first 3 months following aneurysm rupture than those without the allele. There was a trend for this SNP to be associated with increased risk of mortality at the 6-month outcome measurement as well (p = .058). These subjects were 4.137 times (95% CI [0.955, 17.918]) more likely to die within the first 6 months following aneurysm rupture. This finding supports the hypothesis that the ETB gene plays a part in mortality following aSAH.

One of the limitations to this study was small sample size. A larger sample size is preferred in association studies. We continue to recruit at the study site to increase the number of subjects with aSAH. A second limitation is the lack of minorities in our sample. We did not present minority subjects in this study because the size of the minority sample available to us would make it a very underpowered analysis. The minority population is small in the Pittsburgh region and therefore our minority sample is less than 10% of the overall aSAH population. In the future, as we are able to recruit a larger number of minorities, we will be able to report the genetic characteristics of the ET-1 SNPs for this aSAH population.

Another limitation is that patients with a cerebral angiography may have had this procedure due to a worsening in their clinical assessment (e.g., change in level of consciousness). By including patients that have a need for angiography, we skew the sample toward subjects that are in a more critical state. Patients that were in a less critical state may have differences in their genotype distributions when compared to the more critical patients. Such differences in genotype distribution may account for differences in the overall state of the patients. In addition, we were only able to include patients that had follow-up visits with the neuropsychological technician. In the overall study population, 21% of patients were not available for 3-month follow-up and 24% were not available for 6-month follow-up; thus we could not include them in this sample.

In summary, we found that the variant allele of tagging SNP rs6912834 trended toward an association with an increased incidence of CV following aSAH. This finding suggests that the ET-1 protein does play a role in the development of CV after aSAH. Further investigation is needed to find the functional SNP that is within the region of the ET-1 gene represented by that tagging SNP. In addition, two SNPs of the ETB gene were associated with poor outcomes at 3 months after aneurysm rupture, suggesting that ETB plays a role in both functional outcomes and mortality after aSAH. These findings are of high clinical significance. Using these alleles as a screening tool to identify patients who are at higher risk of death and other poor outcomes may allow nurses and other health professionals to focus resources on these at-risk patients, thus improving these outcomes.

Acknowledgments

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by NIH funding (R01NR004339). In addition, it was supported by grants from Sigma Theta Tau, the International Society of Nurses in Genetics, and the Neuroscience Nursing Foundation.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

  1. Anderson C, Anderson N, Bonita R. Epidemiology of aneurysmal subarachnoid hemorrhage in Australia and New Zealand: Incidence and case fatality from the Australasian Cooperative Research on Subarachnoid Hemorrhage Study (ACROSS). Stroke. 2000;31:1843–1850. doi: 10.1161/01.str.31.8.1843. [DOI] [PubMed] [Google Scholar]
  2. Barden AE, Herbison CE, Beilin LJ, Michael CA, Walters BN, Van Bockxmeer FM. Association between the endothelin-1 gene Lys198Asn polymorphism blood pressure and plasma endothelin-1 levels in normal and pre-eclamptic pregnancy. Journal of Hypertension. 2001;19:1775–1782. doi: 10.1097/00004872-200110000-00011. [DOI] [PubMed] [Google Scholar]
  3. Benjafield AV, Katyk K, Morris BJ. Association of EDNRA, but not WNK4 or FKBP1B, polymorphisms with essential hypertension. Clinical Genetics. 2003;64:433–438. doi: 10.1034/j.1399-0004.2003.00148.x. [DOI] [PubMed] [Google Scholar]
  4. Cohen RA, Vanhoutte PM. Endothelium-dependent hyperpolarization: Beyond nitric oxide and cyclic GMP. Circulation. 1995;92:3337–3349. doi: 10.1161/01.cir.92.11.3337. [DOI] [PubMed] [Google Scholar]
  5. Conway JE, Tamargo RJ. Cocaine use is an independent risk factor for cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Stroke. 2001;32:2338–2343. doi: 10.1161/hs1001.097041. [DOI] [PubMed] [Google Scholar]
  6. Crago EA, Thampatty BP, Sherwood PR, Kuo CW, Bender C, Balzer J, Poloyac SM. CSF 20-HETE is associated with delayed cerebral ischemia and poor outcomes after aneurysmal subarachnoid hemorrhage. Stroke. 2011;42:1872–1877. doi: 10.1161/STROKEAHA.110.605816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dorsch NW. Therapeutic approaches to vasospasm in subarachnoid hemorrhage. Current Opinion in Critical Care. 2002;8:128–133. doi: 10.1097/00075198-200204000-00007. [DOI] [PubMed] [Google Scholar]
  8. Gennarelli TA, Spielman GM, Langfitt TW, Gildenberg PL, Harrington T, Jane JA, Pitts LH. Influence of the type of intracranial lesion on outcome from severe head injury. Journal of Neurosurgery. 1982;56:26–32. doi: 10.3171/jns.1982.56.1.0026. [DOI] [PubMed] [Google Scholar]
  9. Gormley K, Bevan S, Hassan A, Markus HS. Polymorphisms in genes of the endothelin system and cerebral small-vessel disease. Stroke. 2005;36:1656–1660. doi: 10.1161/01.STR.0000173173.38289.69. [DOI] [PubMed] [Google Scholar]
  10. Goto Y, Yamagata S. Deteriorating factors on the outcome in patients with fair grade of subarachnoid hemorrhage (WFNS grade I and II). No Shinkei Geka—Neurological Surgery. 2006;34:577–582. [PubMed] [Google Scholar]
  11. Hatake K, Wakabayashi I, Kakishita E, Hishida S. Impairment of endothelium-dependent relaxation in human basilar artery after subarachnoid hemorrhage. Stroke. 1992;23:1111–1116. doi: 10.1161/01.str.23.8.1111. discussion 1116–1117. [DOI] [PubMed] [Google Scholar]
  12. Herrmann S, Schmidt-Petersen K, Pfeifer J, Perrot A, Bit-Avragim N, Eichhorn C, Osterziel KJ. A polymorphism in the endothelin-A receptor gene predicts survival in patients with idiopathic dilated cardiomyopathy. European Heart Journal. 2001;22:1948–1953. doi: 10.1053/euhj.2001.2626. [DOI] [PubMed] [Google Scholar]
  13. Iemitsu M, Maeda S, Otsuki T, Sugawara J, Tanabe T, Jesmin S, Matsuda M. Polymorphism in endothelin-related genes limits exercise-induced decreases in arterial stiffness in older subjects. Hypertension. 2006;47:928–936. doi: 10.1161/01.HYP.0000217520.44176.73. [DOI] [PubMed] [Google Scholar]
  14. Iglarz M, Benessiano J, Philip I, Vuillaumier-Barrot S, Lasocki S, Hvass U, Henrion D. Preproendothelin-1 gene polymorphism is related to a change in vascular reactivity in the human mammary artery in vitro. Hypertension. 2002;39:209–213. doi: 10.1161/hy0202.103442. [DOI] [PubMed] [Google Scholar]
  15. Jennett B. Outcome after severe head injury: Definitions and predictions. Medical Journal of Australia. 1976;2:475–477. [PubMed] [Google Scholar]
  16. Jennett B, Bond M. Assessment of outcome after severe brain damage. Lancet. 1975;1:480–484. doi: 10.1016/s0140-6736(75)92830-5. [DOI] [PubMed] [Google Scholar]
  17. Kasuya H, Weir BK, Nakane M, Pollock JS, Johns L, Marton LS, Stefansson K. Nitric oxide synthase and guanylate cyclase levels in canine basilar artery after subarachnoid hemorrhage. Journal of Neurosurgery. 1995;82:250–255. doi: 10.3171/jns.1995.82.2.0250. [DOI] [PubMed] [Google Scholar]
  18. Kessler IM, Pacheco YG, Lozzi SP, de Araujo AS, Jr., Onishi FJ. Endothelin-1 levels in plasma and cerebrospinal fluid of patients with cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Surgical Neurology. 2005;64S1:2–5. doi: 10.1016/j.surneu.2005.04.014. discussion S1:5. [DOI] [PubMed] [Google Scholar]
  19. Kessler IM, Pacheco YG, Lozzi SP, de Araujo AS, Jr., Onishi FJ, de Mello PA. Endothelin-1 levels in plasma and cerebrospinal fluid of patients with cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Surgical Neurology. 2005;64S1:2–5. doi: 10.1016/j.surneu.2005.04.014. discussion S1:5. [DOI] [PubMed] [Google Scholar]
  20. Kim P, Schini VB, Sundt TM, Jr., Vanhoutte PM. Reduced production of cGMP underlies the loss of endothelium-dependent relaxations in the canine basilar artery after subarachnoid hemorrhage. Circulation Research. 1992;70:248–256. doi: 10.1161/01.res.70.2.248. [DOI] [PubMed] [Google Scholar]
  21. Maas AI, Braakman R, Schouten HJ, Minderhoud JM, van Zomeren AH. Agreement between physicians on assessment of outcome following severe head injury. Journal of Neurosurgery. 1983;58:321–325. doi: 10.3171/jns.1983.58.3.0321. [DOI] [PubMed] [Google Scholar]
  22. Mascia L, Fedorko L, Stewart DJ, Mohamed F, terBrugge K, Ranieri VM, Wallace MC. Temporal relationship between endothelin-1 concentrations and cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage. Stroke. 2001;32:1185–1190. doi: 10.1161/01.str.32.5.1185. [DOI] [PubMed] [Google Scholar]
  23. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research. 1988;16:1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicaud V, Poirier O, Behague I, Herrmann SM, Mallet C, Troesch A, Cambien F. Polymorphisms of the endothelin-A and -B receptor genes in relation to blood pressure and myocardial infarction: the Etude Cas-Temoins sur l'Infarctus du Myocarde Study. American Journal of Hypertension. 1999;12:304–310. doi: 10.1016/s0895-7061(98)00255-6. [DOI] [PubMed] [Google Scholar]
  25. Onoue H, Kaito N, Akiyama M, Tomii M, Tokudome S, Abe T. Altered reactivity of human cerebral arteries after subarachnoid hemorrhage. Journal of Neurosurgery. 1995;83:510–515. doi: 10.3171/jns.1995.83.3.0510. [DOI] [PubMed] [Google Scholar]
  26. Petzold GC, Einhaupl KM, Dirnagl U, Dreier JP. Ischemia triggered by spreading neuronal activation is induced by endothelin-1 and hemoglobin in the subarachnoid space. Annals of Neurology. 2003;54:591–598. doi: 10.1002/ana.10723. [DOI] [PubMed] [Google Scholar]
  27. Qureshi AI, Suri MF, Yahia AM, Suarez JI, Guterman LR, Hopkins LN, Tamargo RJ. Risk factors for subarachnoid hemorrhage. Neurosurgery. 2001;49:607–612. doi: 10.1097/00006123-200109000-00014. discussion 612-603. [DOI] [PubMed] [Google Scholar]
  28. Ratsep T, Asser T. Cerebral hemodynamic impairment after aneurysmal subarachnoid hemorrhage as evaluated using transcranial Doppler ultrasonography: Relationship to delayed cerebral ischemia and clinical outcome. Journal of Neurosurgery. 2001;95:393–401. doi: 10.3171/jns.2001.95.3.0393. [DOI] [PubMed] [Google Scholar]
  29. Roger VL, Go AS, Lloyd-Jones DM, Adams RJ, Berry JD, Brown TM, Wylie-Rosett J. Heart disease and stroke statistics—2011 update: A report from the American Heart Association. Circulation. 2011;123:e18–e209. doi: 10.1161/CIR.0b013e3182009701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seifert V, Loffler BM, Zimmermann M, Roux S, Stolke D. Endothelin concentrations in patients with aneurysmal subarachnoid hemorrhage: Correlation with cerebral vasospasm, delayed ischemic neurological deficits, and volume of hematoma. Journal of Neurosurgery. 1995;82:55–62. doi: 10.3171/jns.1995.82.1.0055. [DOI] [PubMed] [Google Scholar]
  31. Sloan MA, Haley EC, Jr., Kassell NF, Henry ML, Stewart SR, Beskin RR, Torner JC. Sensitivity and specificity of transcranial Doppler ultrasonography in the diagnosis of vasospasm following subarachnoid hemorrhage. Neurology. 1989;39:1514–1518. doi: 10.1212/wnl.39.11.1514. [DOI] [PubMed] [Google Scholar]
  32. Sobey CG, Heistad DD, Faraci FM. Effect of subarachnoid hemorrhage on dilatation of rat basilar artery in vivo. American Journal of Physiology. 1996;271:H126–H132. doi: 10.1152/ajpheart.1996.271.1.H126. [DOI] [PubMed] [Google Scholar]
  33. Spilker J, Kongable G, Barch C, Braimah J, Brattina P, Daley S, Sailor S. Using the NIH Stroke Scale to assess stroke patients. The NINDS rt-PA Stroke Study Group. Journal of Neuroscience Nursing. 1997;29:384–392. doi: 10.1097/01376517-199712000-00008. [DOI] [PubMed] [Google Scholar]
  34. Stegmayr B, Eriksson M, Asplund K. Declining mortality from subarachnoid hemorrhage: Changes in incidence and case fatality from 1985 through 2000. Stroke. 2004;35:2059–2063. doi: 10.1161/01.STR.0000138451.07853.b6. [DOI] [PubMed] [Google Scholar]
  35. Stevens PA, Brown MJ. Genetic variability of the ET-1 and the ETA receptor genes in essential hypertension. Journal of Cardiovascular Pharmacology. 1995;26:S9–S12. [PubMed] [Google Scholar]
  36. Sulter G, Steen C, De Keyser J. Use of the Barthel index and modified Rankin scale in acute stroke trials. Stroke. 1999;30:1538–1541. doi: 10.1161/01.str.30.8.1538. [DOI] [PubMed] [Google Scholar]
  37. Suzuki H, Sato S, Suzuki Y, Oka M, Tsuchiya T, Iino I, Shimoda S. Endothelin immunoreactivity in cerebrospinal fluid of patients with subarachnoid haemorrhage. Annals of Medicine. 1990;22:233–236. doi: 10.3109/07853899009148932. [DOI] [PubMed] [Google Scholar]
  38. Suzuki H, Sato S, Suzuki Y, Takekoshi K, Ishihara N, Shimoda S. Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage. Acta Neurologica Scandinavica. 1990;81:553–554. doi: 10.1111/j.1600-0404.1990.tb01018.x. [DOI] [PubMed] [Google Scholar]
  39. Thampatty BP, Sherwood PR, Gallek MJ, Crago EA, Ren D, Hricik AJ, Poloyac SM. Role of endothelin-1 in human aneurysmal subarachnoid hemorrhage: Associations with vasospasm and delayed cerebral ischemia. Neurocritical Care. 2011;15:19–27. doi: 10.1007/s12028-011-9508-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Treggiari-Venzi MM, Suter PM, Romand JA. Review of medical prevention of vasospasm after aneurysmal subarachnoid hemorrhage: A problem of neurointensive care. Neurosurgery. 2001;48:249–261. doi: 10.1097/00006123-200102000-00001. discussion 261–242. [DOI] [PubMed] [Google Scholar]
  41. Tregouet DA, Garelle V. A new JAVA interface implementation of THESIAS: Testing haplotype effects in association studies. Bioinformatics. 2007;23:1038–1039. doi: 10.1093/bioinformatics/btm058. [DOI] [PubMed] [Google Scholar]
  42. van Gijn J, Kerr RS, Rinkel GJ. Subarachnoid haemorrhage. Lancet. 2007;369:306–318. doi: 10.1016/S0140-6736(07)60153-6. [DOI] [PubMed] [Google Scholar]
  43. Vergouwen MD, Vermeulen M, van Gijn J, Rinkel GJ, Wijdicks EF, Muizelaar JP, Roos YB. Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: Proposal of a multidisciplinary research group. Stroke. 2010;41:2391–2395. doi: 10.1161/STROKEAHA.110.589275. [DOI] [PubMed] [Google Scholar]
  44. Wilson JT, Pettigrew LE, Teasdale GM. Structured interviews for the Glasgow Outcome scale and the extended Glasgow Outcome scale: Guidelines for their use. Journal of Neurotrauma. 1998;15:573–585. doi: 10.1089/neu.1998.15.573. [DOI] [PubMed] [Google Scholar]
  45. Yamaji T, Johshita H, Ishibashi M, Takaku F, Ohno H, Suzuki N, Fujino M. Endothelin family in human plasma and cerebrospinal fluid. Journal of Clinical Endocrinology & Metabolism. 1990;71:1611–1615. doi: 10.1210/jcem-71-6-1611. [DOI] [PubMed] [Google Scholar]
  46. Yamamoto S, Nishizawa S, Yokoyama T, Ryu H, Uemura K. Subarachnoid hemorrhage impairs cerebral blood flow response to nitric oxide but not to cyclic GMP in large cerebral arteries. Brain Research. 1997;757:1–9. doi: 10.1016/s0006-8993(96)01433-3. [DOI] [PubMed] [Google Scholar]
  47. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411–415. doi: 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]

RESOURCES