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. Author manuscript; available in PMC: 2009 Sep 16.
Published in final edited form as: J Cereb Blood Flow Metab. 2008 Mar 5;28(6):1204–1211. doi: 10.1038/jcbfm.2008.11

Endothelial Nitric Oxide Synthase Gene Single Nucleotide Polymorphism Predicts Cerebral Vasospasm following Aneurysmal Subarachnoid Hemorrhage

Robert M Starke 1,2,*, Grace H Kim 1,*, Ricardo J Komotar 1, Zachary L Hickman, Eric M Black 1, Maritza B Rosales 4, Christopher P Kellner 1, David K Hahn 1, Marc L Otten 1, John Edwards 4, Tao Wang 3, James J Russo 4, Stephan A Mayer 1,5, E Sander Connolly Jr 1
PMCID: PMC2744963  NIHMSID: NIHMS106138  PMID: 18319732

Summary

Vasospasm is a major cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (aSAH). Studies have demonstrated a link between single nucleotide polymorphisms (SNP) in the endothelial nitric oxide synthase (eNOS) gene and the incidence of coronary spasm and aneurysms. Alterations in the eNOS T-786 SNP may lead to an increased risk of post-aSAH cerebral vasospasm. In this prospective clinical study, 77 aSAH patients provided genetic material and were followed for the occurrence of vasospasm. In multivariate logistic regression analysis, genotype was the only factor predictive of vasospasm. The odds ratio for symptomatic vasospasm in patients with one T allele was 3.3 (95% CI 1.1–10.0, p=0.034) and 10.9 for TT. Patients with angiographic spasm were 3.6 times more likely to have a T allele (95% CI 1.3–9.6, p=0.013, TT OR 12.6). Patients with severe vasospasm requiring endovascular therapy were more likely to have a T allele (OR 3.5, 95% CI 1.3–9.5, p=0.016, TT OR 12.0). Patients with the T allele of the eNOS gene are more likely have severe vasospasm. Presence of this genotype may allow the identification of individuals at high risk for post-aSAH vasospasm and lead to early treatment and improved outcome.

Keywords: intracranial aneurysm, polymorphism, endothelial nitric oxide synthase, nitric oxide, subarachnoid hemorrhage, vasospasm

INTRODUCTION

The estimated annual rate of aneurysmal subarachnoid hemorrhage (aSAH) among European and North American populations ranges from 6–8 per 100,000 population (Broderick et al 1993; Linn et al 1996). Approximately 30 % of survivors remain dependent, and only 30 – 45 % return to previous or comparable jobs (Ropper and Zervas 1984). Following aSAH, a major cause of morbidity and mortality is delayed ischemic neurological deterioration secondary to cerebral vasospasm (Bendok et al 1998). Symptomatic vasospasm occurs in 15–65% of patients, is detectable in 30–70% of patients by angiography, and results in infarction in 10–45% of patients (Kassell et al 1985; Lanzino et al 1999; Song et al 2006; Vajkoczy et al 2005; Wurm et al 2004).

In vessel endothelium, nitric oxide (NO) is continuously generated from the endothelial nitric oxide synthase (eNOS) gene to maintain basal vascular tone (Quyyumi et al 1995). Aneurysmal SAH results in alterations of eNOS gene expression and disruption of the balanced regulation of cerebral vascular tone (Khurana et al 2002; Weir and MacDonald 1993). Following aSAH, studies have found abnormal cerebrospinal fluid NO levels in humans (Sadamitsu et al 2001; Woszczyk et al 2003). Furthermore, in dogs and ex vivo in the arteries of humans, adenovirus gene transfter of eNOS has shown to be protective in SAH (Khurana et al 2000; Khurana et al 2002). NO is an inhibitor of inflammation and smooth muscle proliferation, pathological changes that are found in cerebral vasospasm (Dumont et al 2003; Moncada et al 1991). Studies have found an association with eNOS polymorphisms and the formation of intracranial aneurysms (Khurana et al 2004) and coronary spasm, (Yoshimura et al 2000) a process that may be physiologically similar to cerebral vasospasm.

We hypothesize that polymorphisms in the eNOS T-786 SNP are associated with the incidence of post-aSAH cerebral vasospasm. Presence of this allele may allow clinicians to identify high-risk individuals and tailor intensive care management in a patient-specific manner. In this context, the principal aims of our prospective clinical study were to 1) determine if there was an association between the presence of such polymorphisms and the presence of post-aSAH vasospasm; 2) determine if differences in such polymorphisms had an effect on the incidence of cerebral infarction secondary to vasospasm.

MATERIALS and METHODS

Patient Population

Patients presenting to our institution from January 2004 to December 2006 with nontraumatic, CT confirmed SAH were prospectively approached for enrollment into this IRB-approved study. Patients were eligible if they presented within 2 days of SAH. After informed consent, a buccal swab was used to collect cheek cells for DNA analysis. All patients provided samples within 3 days of SAH onset. Patients later found to have non-aneurysmal SAH were excluded from the study.

Management Protocol

A standardized management protocol was used in the management of all aSAH patients including immediate aneurysm repair whenever feasible. Decisions regarding treatment modality were made by the combined teams of open-neurovascular and endovascular surgeons. Vasospasm prophylaxis consisted of nimodipine administration and the active maintenance of central venous pressure greater than 5 mm Hg. Post-operatively, patients developing symptomatic vasospasm were treated with induced hypertension and hypervolemia (central pressure ≥ 8mmHg). ICP elevation above 20 mm Hg was treated aggressively with mannitol and/or hypertonic saline.

Study Outcomes

Patient demographic information, medical/surgical history, and clinical course were obtained at the time of enrollment from family members or by review of the medical record. Patients were prospectively followed for adverse events until hospital discharge. All outcomes were compiled blinded to knowledge of a patient’s genotype.

Symptomatic vasospasm was defined as any change in mental status of greater than 2 points in the Glasgow Coma Scale or any new neurological deficit that could not be explained by other etiology (Pickard et al 1989). Patients suspected of clinical vasospasm underwent physical examination, imaging, and laboratory investigation to rule out other possible causes. Other potential causes of clinical deterioration, such as hydrocephalus, rebleeding, cerebral edema, retraction injury, ventriculitis, metabolic derangements, and seizures were rigorously excluded. Clinical deterioration from symptomatic vasospasm thought to be consistent with vasospasm were treated with hypertensive, hypervolemic, and hemodilutional therapy (HHH) to maintain a hematocrit of approximately 30, systolic blood pressure (SBP) >160 mm Hg, and central venous pressure of >8. When clinical evidence of vasospasm persisted for more than 2 hours despite HHHT, cerebral angiography was used to document vasospasm. Additionally, poor-grade patients and sedated patients received CT or MR perfusion on day 4–8 to assess for vasospasm. Patients with vasospasm on CT or MR perfusion then received angiography. On angiography patients were considered to have vasospasm if there was arterial narrowing compared to normal vessel diameter (mild <30%, moderate 30–60%, severe >60%). Patients with moderate or severe angiographic vasospasm were treated with intra-arterial administration of vasodilators and balloon angioplasty when indicated. Cerebral infarction due to vasospasm was defined as any new lucency on CT or DWI positive signal on MRI in the appropriate vascular territory after exclusion of other potential causes.

Genetic Analysis

All genetic analysis was performed by laboratory personnel blinded to patient identities. Genomic DNA was extracted from buccal swabs using MasterAmp Extraction kits (Epicentre, Madison, WI) and ~50 ng DNA was amplified in 10 μl reactions with primers 5′-GCATGCACTCTGGCCTGAAGT-3′ and 5′-CAGGAAGCTGCCTTCCAGTGC-3′ (500 nM each), 0.25 μl JumpStart RED AccuTaq DNA polymerase (Sigma-Aldrich, St. Louis, MO), 1x AccuTaq reaction buffer and 500 μM dNTPs. PCR cycles consisted of 94°C denaturation for 30 sec, annealing for 30 sec (touchdown from 65°C first 10 cycles, 57°C last 20 cycles) and 72°C extension for 90 sec. After a final 5 min extension, PCR products were treated with ExoSapIT kits (USB, Cleveland, OH), bidirectional sequencing was carried out with the same primers on 20–40 ng PCR products using BigDye Termination v3.1 Cycle Sequencing kits (ABI, Foster City, CA), and sequencing products analyzed on an ABI 3730xl capillary instrument. Sequence traces were aligned and SNPs determined using SeqMan software (DNAStar, Madison, WI).

Statistical Analysis

Data are presented as mean and range for continuous variables, and as frequency for categorical variables. Statistical analyses were carried out using unpaired Student’s t-test, Chi-square, and Fisher’s exact tests, Mantel-Haenszel test for linear association as appropriate. Odds ratios were calculated using univariate logistic regression analysis. Factors predictive of all outcomes of vasospasm in univariate analysis (p<0.10) and age, sex, and gender were entered into a multivariable logistic regression. Race, age, gender, stimulant use, smoking, family history, Hunt and Hess and Fischer grade were also controlled for in a comprehensive multivariable logistic regression analysis to assess the ability of eNOS genotype to predict all outcomes of vasospasm. Conformance with the Hardy-Weinberg equilibrium was tested by Chi-square goodness of fit. P-values of ≤0.05 were considered statistically significant.

RESULTS

Cohort Characteristics

Seventy-seven patients with SAH and confirmed aneurysms by angiography provided consent and genetic material for this study. The eNOS T-786 single nucleotide polymorphism was sequenced in all patients. Genotypes were in accordance with the Hardy-Weinberg equilibrium (p=0.46). Minor allele frequencies were found to be similar to those in previously published studies (Akagawa et al 2005; Khurana et al 2004; Krex et al 2006; Krischek et al 2006; Song et al 2006). No significant differences were found in baseline characteristics or treatment between patients according to genotype (see Table 1). Eighteen percent of patients had a family history of intracranial aneurysm.

Table 1.

Distribution of patient characteristics by genotype

CC 7 CT 37 TT 33 Significance p-value
Mean Age, yrs 57±12 54±11 54±12 0.832
Female Sex 5 (71%) 23 (62%) 21 (64%) 0.897
Medical History
Smokers 4 (57%) 24 (65%) 15 (46%) 0.263
Stimulant Use 0 4 (11%) 2 (6%) 0.549
Hypertension 4 (57%) 22 (60%) 21 (64%) 0.915
Diabetes Mellitus 1 (14%) 7 (19%) 1 (3%) 0.116
Relative with an
Aneurysm 1 (14%) 5 (14%) 8 (24%) 0.49
Ethnicity
 White 4 (57%) 19 (51%) 13 (39%) 0.483
 African American 0 3 (8%) 2 (6%)
 Hispanic 0 9 (24%) 10 (30%)
 Other or Unknown 3 (43%) 6 (16%) 8 (24%)
Hunt Hess
 1 1 (14%) 10 (27%) 9 (27%) 0.594
 2 3 (43%) 13 (35%) 8 (24%)
 3 3(43%) 8 (22%) 9 (27%)
 4 0 5 (13%) 3 (9%)
 5 0 1 (3%) 4 (12%)
Fisher Grade
 2 4 (57%) 20 (54%) 11 (33%) 0.313
 3 3 (43%) 14 (38%) 16 (49%)
 4 0 3 (8%) 6 (18%)
Interventions
 Clip 6 (86%) 23 (70%) 25 (83%) 0.371
 Coil 1 (14%) 10 (30%) 5 (17%)
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Cerebral Vasospasm

In total, 43% of patients had either symptomatic or angiographic vasospasm. Thirty percent of patients had symptomatic vasospasm, 38% had angiographic vasospasm, and 35% had vasospasm necessitating endovascular treatment. Twenty percent of patients had an infarction following aneurysm rupture and 10% had infarction secondary to vasospasm.

There was a significant increase in symptomatic or angiographic vasospasm in patients with the TT genotype (61%) versus the CT (35%) or CC genotype (0%, p=0.006, see Table 2). In univariate analysis Fisher grade and eNOS genotype were the only factors predictive of vasospasm. In univariate analysis, the odds ratio (OR) of developing vasospasm in patients with a T allele was 3.2 (95% CI 1.4–7.3, p=0.004, TT genotype OR 10.2). There was a significant increase in angiographic vasospasm and vasospasm necessitating endovascular therapy in the TT (55% and 52% respectively) versus the CT (30% and 27%) or CC polymorphism (0% and 0%, p=0.01 and p=0.013, respectively). In univariate analysis patients with a T had 3.6-fold higher odds of having angiographic vasospasm (95% CI 1.5–8.6, p=0.003, TT genotype OR 13.0), and an OR of 3.6 for requiring endovascular treatment of vasospam (95% CI 1.5–8.8, p=0.004, TT genotype OR 13.0). Forty-two percent of patients with TT, 27% of patients with CT, and 0% of patients with CC had symptomatic vasospasm (p = 0.067). In univariate analysis the OR for symptomatic vasospasm for those patients with the T allele was 2.7 (95% CI 1.12–6.3, p=0.02, TT OR 7.3). In univariate analysis Fisher grade was the only other variable predictive of vasospasm. Using Fisher grade 2 as a reference point, patients with Fisher grade 3 had a 2.0 fold increased risk of vasospasm (95% CI 1.0–4.0, P=0.044). There was no significant increased risk in Fisher grade 4 patients.

Table 2.

Outcome according to genotype

Outcome CC CT TT p-value*
Total 7 37 33
Any Vasospasm 0 13 (35%) 20 (61%) 0.003
Symptomatic Vasospasm 0 10 (27%) 14 (42%) 0.024
Angiographic Spasm 0 11 (30%) 18 (55%) 0.003
Endovascularly Treated Spasm 0 10 (27%) 17 (52%) 0.003
Overall Infarction 1 (14%) 5 (14%) 9 (27%) 0.19
Infarction due to Vasospasm 0 4 (11%) 4 (12%) 0.52
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*

Significance by the Mantel-Haenszel test for linear association

In multivariate logistic regression analysis, genotype was the only significant predictor of vasospasm (see Table 3). The T allele was a significant predictor of vasospasm (OR 3.4 95% CI 1.3–9.0, p=0.013, TT OR 11.6), symptomatic vasospasm (OR 3.3 95% CI 1.1–10.0, p=0.034, TT OR 10.9), angiographic vasospasm (OR 3.6 95% CI 1.3–9.6, p=0.013, TT OR 12.6), and vasospasm necessitating endovascular treatment (3.5 95% CI 1.3–9.5, p=0.016, TT OR 12.0). When patients were stratified according to race in multivariable analysis, Caucasians with a T allele had 3 times higher odds of having symptomatic spasm (p=0.07), a 2.6 times higher odds of angiographic vasospasm (p=0.10), and 2.7 times higher odds of severe vasospasm requiring endovascular treatment (p=0.10). In Hispanic patients with a T allele, the OR of having any vasospasm, angiographic vasospasm, or vasospasm requiring endovascular treatment was significant (12.3, 95% CI 1.3–195, p=0.03; 16, 95% CI 1.3–195, p=0.03; 16, 95% CI 1.3–195, p=0.03). No alleles were significantly associated with vasospasm when subdivided by other ethnicities (p=0.15).

Table 3.

Multivariable logistic regression analysis of genotype as a predictor of vasospasm

Outcome OR for one T allele1 95% CI p-value
Any Vasospasm 3.4 1.29–9.01 0.013
Symptomatic Vasospasm 3.3 1.09–9.97 0.034
Angiographic Spasm 3.6 1.31–9.57 0.013
Endovascularly Treated Spasm 3.5 1.26–9.54 0.016
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1

Genotype was the only significant predictor of vasospasm in multivariate logistic regression analysis when controlling for race, age, and gender. Significance was unchanged when controlling for race, age, gender, stimulant use, smoking, family history, Hunt and Hess and Fisher grade.

Infarction

There was no significant difference in infarction in patients according to genotype. Fourteen percent of patients with CC and 14% of patients with CT had infarction versus 27% of TT patients (p=0.327). Zero patients with CC had infarction due to vasospasm versus 11% and 12% of patients with CT and TT respectively (CC versus CT or TT, p=0.345).

DISCUSSION

Numerous studies have attempted to determine prognostic indicators of vasospasm as identification of high-risk individuals may allow post-aSAH management to be tailored in a patient specific manner. History of smoking, preexisting hypertension, and hypovolemia have all been linked with vasospasm (Lasner et al 1997; Rabinstein 2006). While experts have argued over the role of age, the strongest known predictive factor for vasospasm is the amount of blood on CT (Fisher grade) which often correlates with the severity of vasospasm (Davis et al 1980; Fox and Ko 1978). Differences in incidence of vasospasm and outcomes in patients with similar Fisher grade, and a genetic association with aneurysm formation has led to the theory that genetic polymorphisms encoding vascular regulatory proteins may result in variable levels of vascular spasticity in response to aSAH.

It is likely that the onset of aSAH is multifactorial in nature and dependent on genetic characteristics and clinical variables associated with aneurysm rupture. The eNOS T-786 SNP has received much attention due to its location within the promoter region of the nitric oxide gene (Nakayama et al 1999). In this study Fisher grade and genotype were the only significant predictors of vasospasm in univariate analysis. In multivariable analysis, genotype was the only independent predictor of vasospasm when accounting for race, age, gender, stimulant use, smoking, family history of aneurysm, and Hunt and Hess and Fisher grades. Patients with a T allele had a 3.3 times higher odds of developing symptomatic vasospasm and patients with the TT genotype were 10.9 times more likely. Patients with the T allele had an OR of 3.5 of necessitating treatment for vasospasm and patients with a TT genotype had an OR of 12.0; thus, the T allele is not only a predictive marker of vasospasm, but a risk factor for severe vasospasm requiring endovascular therapy.

When stratified according to genotype, there was no significant difference in the incidence of infarction due to vasospasm. Twelve percent of patients with the TT and 11% of patients with the CT genotype had infarction due to vasospasm versus 0 in patients with the CC genotype. Although this difference was not statistically significant, our study was not powered to detect differences in infarction. This may also be due to a large number of clinical and genetic factors that link vasospasm with infarction in a vascular teritory. This may also be because we treat vasospasm aggresively with the intent to avoid the development of infaction.

In this study all polymorphisms were in Hardy Weinberg equilibrium. Minor allele frequencies were not significantly different from those in previous studies, (Akagawa et al 2005; Khurana et al 2004; Krex et al 2006; Krischek et al 2006; Song et al 2006) and there was no significant difference in racial groups according to genotype. These results are consistent with more recent studies in larger populations (Rossi et al 2006). Earlier, smaller studies hypothesized that the CC genotype was due to new mutaions as the incidence of CC was 0.9% (n=335), (Nakayama et al 1999) but recent studies in larger populations (n=1096) have found the CC allele to be in Hardy Weinberg equilibrium (19.2%) (Rossi et al 2006). In multivariate logistic regression analysis when controlling for clinical variables, eNOS genotype was the only significant predictor of all forms of vasospasm. There was a trend in Caucasians with a T allele to have an increased risk of symptomatic vasospasm (OR 3, p=0.07) and severe vasospasm requiring endovascular treatment (OR 2.7, p=0.10). The T allele was not significantly associated with an increased risk of all types of vasospasm in each racial subgroup, but this may have been due to the small numbers of patients in ethnic subgroup analysis.

Our results contrast to a previous study examining genetic polymorphisms of eNOS T-786 SNP and cerebral vasospasm. This may be due to inadequate power, and/or differences in ethnicity, incidence of alleles, and prevalence of vasospasm. In one study of 28 Fisher grade III aSAH patients, they found that patients with vasospasm had a nonsignificant increased risk of having had the C allele (odds ratio 7.1 95% CI 0.88–57.5) (Khurana et al 2004). These results may have been due to underpowering or due to the fact that only one of these patients had the CC genotype. In a study of vasospasm following aSAH in 133 Korean patients, the eNOS T-786C polymorphism did not confer any increased risk of symptomatic vasospasm, but the CT genotype was associated with worse outcome (Song et al 2006). These differences may again be accounted for by the variance in racial characteristics; zero Korean patients had the CC genotype. They also may be accounted for by the low rate of symptomatic vasospasm (12.8%) seen in this Korean population as compared to previously published studies (20–65%) (Kassell et al 1985; Lanzino et al 1999; Song et al 2006; Vajkoczy et al 2005).

Although nitric oxide plays a role in vasospasm following aSAH, the precise pathophysiology remains unclear. Studies have proposed that this process may be due to increased (Ignarro 1990; Pluta et al 1996) or decreased nitric oxide levels (Dumont et al 2003; Moncada et al 1991). Studies have reported decreased levels of nitric oxide in animals following experimental aSAH (Kasuya et al 1995); other studies in humans with aSAH found an initial decrease in nitric oxide followed by an increase in NO between 2–8 days (Woszczyk et al 2003).

It is unclear how different allelic combinations of the eNOS T-786 SNP lead to pathological processes. Different allelic combinations have been associated with coronary spasm (CC), (Nakayama et al 1999) aneurysm size (CT), (Akagawa et al 2005) and outcomes following coronary spasm (CT), (Nishijima et al 2007) cerebral vasospasm (CT) (Song et al 2006) and cardiovascular mortality (TT) (Rossi et al 2006). The role of eNOS expression in aSAH is controversial (Pluta et al 1996), (Kasuya et al 1995), (Hino et al 1996; Park et al 2001), (McGirt et al 2002). Pathogenesis due to alterations in polymorphisms may occur through a variety of mechanisms including decreased promoter activity leading to decreased nitric oxide or increase promoter activity leading to increased nitric oxide. Increased nitric oxid may lead to oxidative stress (Yung et al 2006), intimal hyperplasia (Hingorani et al 1999; Kuhlencordt et al 2001), systemic hypertension (Kuhlencordt et al 2001), vascular smooth muscle proliferation (Dumont et al 2003; Moncada et al 1991), increased platelet aggregation, and pro-inflammatory adhesion (Dumont et al 2003; Moncada et al 1991). These mechanisms of dysfunction may lead to alterations in smooth muscle proliferation, vessel dilation and inflammation seen in the pathogenesis of vasospasm following aSAH (Dumont et al 2003; Khurana et al 2004; Moncada et al 1991).

In one study, the eNOS promoter was isolated from patients with coronary spasm, and after being subjected to hypoxic conditions for 24 hours, it was found that genes with the C allele had decreased promoter activity (Nakayama et al 1999). In this study of Asian patients, only 3 of 335 patients had the CC genotype which differs significantly from other studies of Caucasian patients where the CC genotype comprised 19% of 1096 patients (Rossi et al 2006). In this larger study by Rossi et al. patients with the CC genotype had significantly higher levels of nitrotyrosine, a marker of nitric oxide production, thus supporting the notion that patients with the TT genotype may produce lower levels of NO. Unlike coronary spasm, most patients with aSAH do not experience cerebral vasospasm until 3–14 days after aSAH and the role of polymorphisms, the eNOS promoter, and nitric oxide levels have yet to be elucidated over this time period (Pluta et al 1996), (Kasuya et al 1995), (Hino et al 1996; Kassell et al 1984; McGirt et al 2002; Park et al 2001).

Further studies of eNOS, the promoter, and polymorphisms are needed to develop therapeutic agents that target the NO cascade. Preliminary studies have begun to look at invasive techniques to administer exogenous NO (Afshar et al 1995; Wolf et al 1998) or L-arginine (an NO substrate) (Nakaki et al 1990; Pluta et al 2000) in the treatment of vasospasm. Adenovirus gene transfter of eNOS to human arteries ex vivo and canine arteries in vivo has been shown to be protective in SAH models (Khurana et al 2000; Khurana et al 2002). Statins have recently been found to decrease vasospasm following aSAH. The presumed mechanism is the ability of statins to upregulate eNOS or to decrease reactive oxygen free radicals, platelet aggregation, and inflammation that leads to endothelial damage caused by blood clots (Tseng et al 2005). Such interventions may decrease the significant morbidity and mortality due to vasospasm, as well as decrease complications due to triple therapy, angiography, and endovascular treatment of vasospasm.

Future studies should focus on the link between vasospasm, eNOS 786 polymorphisms, the eNOS promoter and the amount of nitric oxide produced from the time of aSAH to the end of the vasospasm window (day 14). The role of the eNOS promoter and the corresponding levels of nitric oxide following aSAH throughout the window of vasospasm also needs to be elucidated. These results should be correlated with long term outcomes as it is unclear as to whether decreased incidence of vasospasm will result in improved functional outcome. The results of this study should be confirmed in larger racially diverse subgroups.

CONCLUSION

In conclusion, patients with a T allele have a significantly increased risk of developing severe vasospasm. This may help clinicians predict and identify high risk patients. Furthermore, genetic analysis may allow clinicians to optimize early treatment for patients at risk for vasospasm and infarction and improve patient outcomes.

Acknowledgments

We would like to thank Lina Bruno for helping to bring this project together. RMS was supported in part by an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship. RMS was also partially supported by the CTSA Grant UL1 RR025750 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NCRR or NIH.

Footnotes

Disclosure/Conflict of Interest

The authors have no personal or institutional financial interest in drugs or materials in relation to this paper.

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