amin kassam, m.d., yue-fang chang, ph.d., robert e. ferrell,...

A Functional Polymorphism in the Endothelial Nitric Oxide Synthase Gene is a Risk Factor for

Subarachnoid Hemorrhage in Normotensive Patients with Intracranial Aneurysms

Amin Kassam, M.D., Yue-Fang Chang, Ph.D., Robert E. Ferrell, Ph.D. and David G Peters, Ph.D.

From the Department of Neurosurgery, School of Medicine (A.B.K., Y.C.) and Department of Human Genetics, Graduate School of Public Health (R.E.F., D.G.P.), University of Pittsburgh.

Amin B. Kassam

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Correspondence author:

David G. Peters Ph.D.

Department of Human Genetics

A300 Crabtree Hall

University of Pittsburgh

130 DeSoto St

Pittsburgh, PA 15213

Tel No. 412-624 3018

Fax. No. 412-624 3020

Email: [email protected]

Amin B. Kassam

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ACKNOWLEDGMENT

This work was supported by the Copeland Foundation of Pittsburgh (ABK, DGP) and NASA (NCCI-1227)

(DGP).

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Abstract

Background and Objective: Hypertension and smoking are considered to be important environmental factors

associated with the formation and rupture of intracranial aneurysms (ICA). Recently, hemodynamic forces

have also been implicated as critical intrinsic factors associated with the pathogenesis of ICAs. Nitric oxide

is a potent vasodilator that has been shown to play an important role in vascular homeostasis, and the

expression of endothelial nitric oxide synthase (NOS3) is regulated by hemodynamic shear stress. The

purpose of this study was to determine whether two functional polymorphisms in the NOS3 gene are

associated with presence and/or rupture of ICAs.

Methods. Ninety-seven ICA cases and 187 control volunteers were genotyped for the T-786 C functional

polymorphism in the NOS3 promoter and the conservative Glu298Asp polymorphism within exon 7.

Comparison was made amongst ICA patients and these controls as well as internally within ICA patients

following a series of sequential stratifications that were determined a priori.

Results. The comparison among the ICAs patients and controls was limited by differences in the population,

but not withstanding this no differences in frequency distribution of any of the NOS3 polymorphisms

examined were found. The comparison within aneurysm patients only, without stratification for smoking

and hypertension, showed no association between NOS3 genotype and incidence or rupture of ICA.

However, after stratifying the analyses for hypertension, we found a significant association between exon7

genotype and rupture status in normotensive patients OR=0.16, 95%CI=(0.04, 0.65). Specifically, among

normotensive individuals, 80% of patients with an unruptured ICA were GG (Glu298) homozygotes as

opposed to only 38.2% of patients with ruptured ICA. Similarly, a marginal association between rupture

status and exon7 genotype was observed among smokers OR = 0.25, 95%CI = (0.06, 1.04). Specifically,

among smokers, 81.3% of patients with an unruptured ICA were GG homozygotes as opposed to only 51.6%

of patients with ruptured ICA.

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Conclusions. This preliminary analysis suggests that a functional polymorphism in exon7 of the NOS3 gene

is protective against ICA rupture in normotensive patients and smokers. These data raise the possibility that

NOS3 genotype has potential as an independent predictive marker in diagnosis and management of

aneurysmal disease.

Running title: eNOS a Risk Factor of SAH

Key words: aneurysms, genotype, modeling, NOS3, polymorphism, subarachnoid hemorrhage

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Introduction

Over the past decade significant effort has been invested in trying to gain a better understanding of

the pathogenesis and natural history of intracranial cerebral aneurysms (ICAs). This has been partly a

reflection of the dramatically different outcomes associated with the management of ruptured and unruptured

aneurysms. In the case of unruptured aneurysms, the operative mortality has been reported to be as low as

2.5% and morbidity less than 6% (13-16,30). However, once an aneurysm ruptures subarachnoid

hemorrhage (SAH) (43) occurs and the impact becomes much more devastating (32). It would be of great

value to identify risk factors that could predict growth and rupture of ICAs. To date much of the work has

focused on the identification of environmental factors with cigarette smoking (1,17,22) and hypertension

(19,34,44) showing the most consistent associations with aneurysm formation and rupture.

There is now considerable evidence suggesting that ICAs have a significant genetic component. For

example, a ruptured ICA confers increased risk of ICA in first degree relatives (33) and a number of groups,

including our own have reported association of ICAs with functional polymorphic sites at the DNA level

(28,37). Based on these findings we sought to examine the possibility of identifying molecular markers that

are associated with arterial homeostasis (arterial wall repair and tear mechanisms) that may serve as markers

predictive of aneurysm growth and rupture. We believed that endothelial nitric oxide synthase (NOS3)

would be a likely candidate based on the role it serves in vascular tone modulation. Endothelial nitric oxide

synthase is a heme-containing enzyme that catalyzes the NADPH- and [O2]-dependent five-electron

oxidation of L-arginine to nitric oxide (NO) and citrulline. (24) NO has a powerful vasodilatory action and

plays a central role in the regulation of vascular resistance (4,21). Other functional consequences of its

production include the suppression of the proliferation of vascular smooth muscle cells (12,31), and

inhibition of the adherence of circulating blood cells (platelets and leukocytes) to the endothelium (29,36).

It has recently been shown that the presence of a T-786 C base substitution/single nucleotide

polymorphism (SNP) in the promoter of the NOS3 gene, results in reduced transcription of NOS3 mRNA

(25). It has also been reported that a Glu298Asp polymorphism in exon 7 results in a reduction in functional

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NOS protein in individuals who are either homozygous or heterozygous for the T allele at this locus (38).

Furthermore both polymorphisms have been shown to be associated with coronary vasospasm (25,45).

We hypothesized that there exists a continuous balance between arterial wall disruption secondary to

the pulsatile effects of hemodynamic forces within the Circle of Willis and the repair of resulting

microinjuries. This natural state of damage and repair (arterial homeostasis) may be disrupted in some

individuals thus predisposing them to intracranial cerebral aneurysms. We further hypothesized that a

genetically programmed reduction in cerebrovascular NO would result in an altered homeostatic response to

the effects of hemodynamic stress. This could exacerbate the disruptive effects of stress on the arterial wall

at bifurcations within the Circle of Willis, thereby increasing the likelihood of arterial injury. Furthermore,

an inappropriate response to arterial injury through increased smooth muscle dedifferentiation, proliferation

and migration and increased leukocyte adherence could result in altered reparative processes resulting in

inappropriate tissue remodeling process and the eventual loss of arterial wall integrity. These processes

might also be deregulated by a reduction in functional cerebrovascular NO. Based on these premises we

sought to determine whether the NOS3 T-786 C or Glu298Asp polymorphisms were associated with either

the incidence of ICA, propensity for ICA rupture, or incidence of multiple aneurysms.

Materials and Methods

Study Population and Data Collection

This research was approved by the Institutional Review Board of the University of Pittsburgh

(#951220) and all participants gave written informed consent. The Study group consisted of ninety-seven

unrelated patients undergoing repair of an intracranial aneurysm at the University of Pittsburgh Medical

Center, Pittsburgh, PA. Initially, baseline demographics, medical and family histories were ascertained

followed by collection of peripheral blood specimens for subsequent DNA extraction (see below) and NOS3

genotyping. The control group was sampled from a series of unrelated individuals recruited and genotyped

for NOS3 in a population-based study of coronary artery disease risk-factors. Geneotype data for NOS3

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were available in 184 and 187 control patients for the T-786 C and the Glu298Asp polymorphisms

respectively. All of the control subjects reside in Western Pennsylvania and are drawn from the same

population base as the study subjects with ICAs. In order to further minimize the potential confounders the

following additional exclusion criteria: prior history of psychiatric illness, hypertension, diabetes or

symptomatic coronary artery disease, elevated cholesterol levels (LDLc > 160 md/dL), extreme leaness or

obesity (BMI < 20 or >34 Kg/m2), excess alcohol consumption (> 5 drinks/day), and in women, hormone

replacement therapy, history of hysterectomy or bilateral oophorectomy.

Based on the existing literature surrounding the environmental risk factors associated with aneurysm

formation and rupture an a priori decision had been made to stratify based on the exposure to cigarette

smoking and chronic hypertension. Specifically, the patients were asked if they currently engage in cigarette

smoking or not. Similarly we considered hypertension as a dichotomous variable. A patient was considered

as hypertensive if they required antihypertensive medication on a chronic basis prior to this hospitalization.

NOS3 Genotyping

High molecular weight genomic DNA for genotyping was prepared by standard methods (23). For

the T-786 C and Glu298Asp SNPs, either a 512 bp or 517 bp segment of the NOS3 gene, respectively, was

amplified by polymerase chain reaction (PCR) using a final concentration of 1 µM each of the following

primers:

5’-CAGATGCCCAGCTAGTG (T-786 C forward)

5’-GGACCTCTAGGGTCATGC (T-786 C reverse)

5'-GACCCTGGAGATGAAGGCAGGAGA (G894 T forward)

5'-ACCACCAGGATGTTGTAGCGG-TGA (G894 T reverse).

PCR was carried out in a total volume of 15 µl using 30 ng genomic DNA in 20 mM Tris-HCl (pH

8.4), 500 mM KCl, 1.5 mM MgCl-2- using 1 unit of Taq polymerase (Life Technologies) and 200 µM each

dNTP. Reactions parameters were as follows: initial denaturation at 95 oC for 4 min, then 30 cycles of

denaturation at 95 oC for 30 s, annealing at 60 oC for 30 s and extension at 72 oC for 30 s. A 5 min chase

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reaction was then carried out at 72 oC. Whole 15 µl reactions were digested by adding 2.75 µl of distilled

water, 2.0 µl of 10 X reaction buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol

[pH 7.9]) and 0.25 µl (5 units) of MspI (NEB). Restriction digestion products were then separated on a 2 %

agarose gel and stained with ethidium bromide and visualized under ultraviolet illumination.

For the T-786 C SNP, Msp-1 digestion of amplified DNA derived from individuals who are

homozygous for the T allele resulted in two DNA fragments of 285 and 221 bp. DNA from heterozygous

individuals yielded four fragments of 285, 221, 177 and 44 bp and individuals homozygous for the C allele

yielded three fragments of 285, 177 and 44 bp. For the Glu298Asp polymorphism, Ban-II digestion of

amplified DNA derived from individuals who are homozygous for the G allele resulted in no cleavage.

DNA from heterozygous individuals yielded three fragments of 517 bp, 346 bp and 171 bp and individuals

homozygous for the T allele yielded two fragments of 346 bp and 171 bp.

Statistical Analyses

Genotype and allele frequencies were estimated by gene counting. The frequencies in all study

groups were compared by standard Chi-square analysis and odds ratio were calculated using SPSS software.

The analysis plan took into consideration the key stratification variables relating to features inherent to the

aneurysm (rupture status and multiplicity) and environmental factors (smoking and hypertension). First an

analysis comparing the entire aneurysm cohort against the controls was performed to assess the association

of ICA propensity based on NOS3 genotype. Then a comparison of just ICA patients was undertaken to

address the association of rupture versus NOS3 genotype.

A baseline analysis was undertaken comparing the aneurysm subjects describing key demographic

and environmental features amongst the ruptured and unruptured patients. Each of the two NOS3 genotypes

was compared by rupture status and then separately by multiplicity of aneurysms at presentation. Once the

aneurysm features were considered the interaction with environmental factors was addressed. In this

analysis the rupture status for each genotype was now stratified by the exposure to each critical

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environmental risk factor (smoking and then hypertension). This was done separately for each genotype and

respective odds ratios were calculated.

Results

Analysis of aneurysm patients vs. controls

Genotype data for the T-786 C polymorphisms were available for 82 subjects with an aneurysm and

for 185 controls. Similarly, the genotype data for the Glu298Asp polymorphisms were available in 96

subjects with an aneurysm and 187 controls. The initial analysis consisted of simply comparing the entire

cohort of aneurysms patients against the entire control group for the frequency of alleles at each polymorphic

site: (1) NOS3 T-786 C (table 1a) and (2) Glu298Asp (table 1b). In this initial analysis the genotype

frequencies did not differ significantly from the expectation of Hardy Weinberg equilibrium. Genotype

frequencies between the aneurysms cases and controls were not significantly different for either the T-786 C

or Glu298Asp polymorphism (P=0.69 and p=0.56 respectively, table 1).

The control group available for comparison significantly limits this analysis. Specifically, while the

demographics of both the study and control groups are similar once restricting for ethnicity, the

environmental factors are more difficult to address. For example, the incidence of smoking was only 9.9%

in the study group when compared to 50.5% in the aneurysm patient group. Similarly hypertension, a key

environmental variable was one of the exclusion criteria in the control group.

Analysis among aneurysm patients

Because of the limitations stated above and an a priori decision to consider stratification by rupture

status an analysis of the aneurysm patients was made. Therefore, the remainder of the analysis was restricted

to this comparison within aneurysm among cases. First, the baseline demographic and environmental risk

factor profiles of the 97 aneurysm subjects were considered and are summarized in table 2. Among these 97

aneurysmal patients we genotyped, 20.6 % were male and 79.4 % were female with an age range of 19-65

(mean 46.6, SD 12.3) and 20-75 (mean 50.7, SD 11.7) respectively. Sixty-three (65.6 %) of the individuals

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presented with SAH due to a ruptured ICA, whereas 33 (34.4 %) underwent elective craniotomy to repair an

unruptured ICA. In one case, the rupture status could not be definitively determined and this patient was

excluded. Fifty-one percent were diagnosed with a single ICA whereas 49 % presented with multiple ICAs.

Twelve percent reported a history of symptomatic ICA among a first or second-degree relative. None had a

personal or family history of connective tissue disorders or autosomal dominant polycystic kidney disease.

Hypertension (defined a baseline chronic blood pressure elevation where the patient required medication at

home for control) was noted in 44.3% of the patients. Smoking status (defined as currently smoking

immediately preceding hospitalization) was available on 89 (92%) of the patients with 50.5% of the total

subjects admitting to smoking cigarettes regularly just prior to their current hospitalization. There were no

statistically significant differences found in the prevalence of medically treated chronic hypertension or

smoking between the ruptured or unruptured patients (Table 2).

Comparison of ruptured and unruptured patients:

The cohort of aneurysms subjects was segregated based on whether the aneurysm was ruptured or

unruptured at the time of presentation and a comparison of the frequency of each NOS3 genotype was made.

There were no statistically significant associations noted between rupture and the presence of the T-786 C

(p=0.62) or Glu298Asp (p=0.22) genotypes (tables 3a and 3b respectively).

Comparison of single or multiple aneurysms at presentation:

Next we compared the aneurysm cohort based on the presence of single versus multiple aneurysms

identified at the time of presentation (Table 4a and 4b). Again we found no difference between subjects

harboring a single aneurysm when compared to those presenting with multiple aneurysms (tables 4a and 4b).

These data indicate that neither the T-786 C nor the Glu298Asp polymorphism in the NOS3 gene is

associated with incidence of ICA, propensity for ICA rupture (not adjusted for smoking or hypertension), or

number of aneurysms.

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Stratification Based on Environmental Factors:

We undertook further stratification analysis in order to take into consideration the potential effect of

each key variable: (1) aneurysm features, i.e., rupture status and (2) environmental factors, i.e., smoking and

then hypertension separately.

Comparison of ruptured vs. unruptured patients by smoking status:

In this analysis the ICA subjects were simultaneously segregated based on rupture status and the

exposure to smoking and then sequentially examined for the frequency distribution of each polymorphism T-

786 C (table 5a) and Glu298Asp (table 5b). This analysis revealed that T-786 C genotype frequencies

between ruptured ICA patients and unruptured ICA patients were not significantly different among smokers

(OR=4.1, 95% C.I.=(0.1,1.5)) and non-smokers (OR=0.7, 95% C.I.=(0.2,3.5)). When the Glu298Asp

genotypes were next examined a marginal significance amongst smokers was noted. Specifically, table 5b

illustrates that when examining the relationship between rupture and Glu298Asp polymorphisms the

genotype GG was identified in 81.3% of unruptured patients in comparison to only 51.6% of ruptured

patients (OR=0.25, 95% C.I.=(0.06,1.04)).

Comparison of ruptured vs. unruptured subjects by hypertension status:

A similar analysis was undertaken for each genotype stratified by hypertension (tables 6a and 6b).

We first considered the T-786 C genotype and again no statistically significant difference was observed in

either the hypertensive (OR=1.21, 95% C.I. (0.3,4.7)) or normotensive patients (OR=0.35, 95% C.I.

(0.1,1.3)). We then examined the distribution of Glu298Asp genotypes in the group stratified by

hypertension and rupture status. As demonstrated in table 6a, hypertensive patients with a ruptured

aneurysm did not have a statistically significant difference in their Glu298Asp genotypes when compared to

unruptured patients (OR=1.02, 95% C.I. (0.3,3.7)). However, when we examined normotensive patients it

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was noted that 80% of the ICA subjects with unruptured aneurysms were of a homozygous GG (Glu298)

geneotype for Glu298Asp; whereas, in comparison only 38.2% of the ruptured aneurysm normotensive

patients were homozygous GG. This difference was statistically significant with an OR=0.16, 95% C.I.

(0.04,0.65) (table 6a and b). Furthermore, the hypertension adjusted odds ratio also reached marginal

significance OR = 0.42, 95%C.I. (0.17, 1.05) with the upper limit just overlapping 1.

Analysis of Allele Frequency

In addition to the above analyses comparing genotype frequencies, we also examined the allele frequency

between the groups. Differences in allele frequency at both loci between the cases and controls were not

significant. Among the cases, the T-786 C allele frequency was also similar regardless of whether the

patient had a ruptured or an unruptured aneurysm. However, among the normotensive aneurysm patients,

the Glu298Asp G allele was less common in the patients with a ruptured aneurysm when compared to the

patients with an unruptured aneurysm (G allele frequency 63.2% vs. 86.7%, p=0.02). This is in keeping with

our analyses of NOS3 genotype.

Discussion

Considerations in Generating a Predictive Model:

It would be of immense value to identify risk factors that are associated with the development and

subsequent rupture of intracranial aneurysms. Clearly the generation of a predictive model will require

multifactorial input variables, likely with differential weighting. Much of the work to date has focused on

environmental variables with smoking and hypertension emerging as the most commonly cited risk factors.

However, there is now considerable evidence suggesting the importance of molecular and genetic variables

that will have to be factored into any biological model developed to predict the natural history of ICAs.

Schievink (33) observed an increased frequency of ICAs among the first degree relatives of ICA

probands and a number of groups have reported genetic association between single candidate genes and the

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occurrence of ICA among populations of unrelated individuals suffering from a seemingly sporadic form of

the disease (28,37). However, apart from a clear association between rare single gene defects that segregate

in a Mendelian fashion in a number of well characterized disorders, such as autosomal dominant polycystic

kidney disease and Ehler’s Danlos Syndrome in which individuals often develop ICA (20,27), there is no

clear pattern of inheritance associated with ICAs. Significant work focusing on linkage analysis is currently

underway to identify the location of genes that contribute to the risk of ICA (The International Familial

Intracranial Aneurysm Study).

To date candidate gene studies to identify genetic risk factors for ICA have focused on genes

involved in extracellular matrix integrity and remodeling. There is good reason for this. Kim et al. (18) and

Bruno et al.(3) studied the biochemistry of ICA domes harvested during elective craniotomy and found

elevated levels of matrix metalloproteases (MMPs) relative to samples of cerebrovasculature from unaffected

controls. The MMPs are critical molecules in the turnover of the extracellular matrix of the arterial wall

responsible for structural stability. Similar studies noted that levels of such enzymes were higher in the

serum of ICA patients than unaffected individuals (40). Furthermore, it has been shown that ICA patients

have lower levels of circulating anti-proteases (11). Accordingly, we have recently shown that genetically

determined differences in levels of proteolytic enzymes and their inhibitors may contribute to the risk of ICA

and have also exluded variation in a number of structural proteins as being associated with ICA (28,35).

Based on the above findings, it is reasonable to assume that the formation and eventual rupture of an

ICA may be the result of a dynamic process of tissue remodeling that is impacted upon by physical and

environmental factors and a genetically predetermined response to these factors at a molecular level. One

such factor is hemodynamic stress. Even under normal circumstances, the arteries in the Circle of Willis

undergo continuous biomechanical insult (8). The ability of the vascular endothelium to perceive the

magnitude of these forces and to respond appropriately is critical to the maintenance of arterial wall integrity

(arterial homeostasis). One attractive hypothesis relating to aneurysm development is, therefore, that at-risk

individuals have an altered/inappropriate homeostatic response to hemodynamic stress, which triggers

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chronic destructive biochemical alterations in the process of extracellular matrix remodeling. When this

process is disrupted the resulting extracellular matrix that provides structural integrity is compromised. This

can create an impaired ability to deal with hemodynamic forces, which if accentuated, may further accelerate

the arterial wall failure. This failure may be progressive along a continuum, initially beginning with altered

ability to maintain vascular tone and the development of a saccular dilatation and then if allowed to progress

culminating in complete homeostatic structural failure with a consequent catastrophic rupture. It is likely

that subtle variation in the ability to respond to these hemodynamic forces is genetically programmed and it

is, therefore, plausible that non-random variation at the DNA level predisposes individuals not only to more

frequent breakdown in the structural integrity of the cerebrovasculature but also to an inefficient and/or

inappropriate response to this vascular injury which may itself be pathogenic, i.e., impaired arterial

homeostasis.

Discussion of Current Study Results:

Based on the discussion above we selected NOS3 as an ideal initial gene to examine for

polymorphisms that may be associated with ICA pathogenesis and rupture as a preliminary step in

developing a predictive model. Specifically, we investigated the association between a T-786 C

polymorphism in the NOS3 promoter and a Glu298Asp polymorphism in exon 7. The selection of these

polymorphisms was based on recent studies demonstrating that the T-786 C SNP modulates the expression

of NOS3 at the level of transcription, the C allele conferring lower levels of promoter activity (25)

Furthermore, it has also been reported that the Glu298Asp polymorphism in exon 7 results in a mature NOS

protein that is susceptible to intracellular cleavage resulting in the generation of N-terminal 35-kDa and C-

terminal 100-kDa fragments (38). As discussed previously, this results in a reduction in functional NOS3

protein in individuals who are either homozygous TT (Asp298) or heterozygous GT (Glu298Asp) at this

locus. We therefore postulated that such a functional impairment associated with the TT homozygous or GT

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heterozygous state would result in impaired homeostasis and predispose patients to the development and

subsequent rupture of ICAs.

In order to assess this vulnerability and subsequent increased frequency of ICA formation based on

NOS3 genotype variations we needed to first consider a non-aneurysmal control group for comparison. The

control group that had NOS3 genotype data available was a part of a population-based study of coronary

artery disease risk factors. While demographically the control group was comparable the key environmental

factors (smoking and hypertension) were distinctly different. As a result this study is limited in being able to

address this first question of increased propensity of ICA formation based on NOS3 genotype variations.

However, this study does have some preliminary value in addressing the next question: what is the potential

effect of NOS3 polymorphisms on the likelihood of rupture? In keeping with the concept of a multifactorial

model the environmental factors need to be considered when trying to answer this question. In examining

the aneurysm study population no difference between exposure to hypertension and smoking amongst

ruptured and unruptured patients was noted (table 2). This is an unexpected finding based on the literature

and may be a reflection of the way in which these two variables were defined. Specifically, no effort was

made to quantitate these variables, but rather, they were considered as dichotomous. It is plausible that

differences may emerge if these were considered as continuous variables. We chose not to do this because

we felt it is unreliable to retrospectively quantify these exposures. This is better addressed in a prospective

study design.

Interestingly, when a more detailed analysis was undertaken adjusting for the effects of smoking and

hypertension in considering the association of rupture and NOS3 genotyping several findings emerged.

A marginal association among smokers, rupture status and Glu298Asp genotype was observed. Specifically,

48.4 % of the smokers that ruptured were found to be either hetereozygous (GT) or homozygous (TT) for the

T allele, whereas, only 18.8% of the unruptured smokers displayed these genotypes. This correlated with an

odds ratio of OR = 0.25, 95%CI = (0.06, 1.04) which just overlaps with 1.

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In order to further understand the interaction of environmental factors and rupture we then considered

the effect of hypertension. The majority of patients who were normotensive and had a ruptured ICA were

found to be homo/hetereozygous for the Glu298Asp T allele. Specifically, 61.8% of the ruptured patients

that were normotensive (not being treated chronically for hypertension) versus 20% of those that were

hypertensive were either Glu298Asp GT or TT (table 6b). The odds ratio proved to be very significant

OR=0.16, 95%CI=(0.04, 0.65). This suggests that there is a protective effect of being homozygous (GG) for

Glu298Asp that is lost in individuals who are hypertensive. Presumably the biological effect of NOS3

variation is outweighed by elevated hemodynamic stress and vascular remodeling secondary to chronic

hypertension. In contrast, there is almost and even distribution of T homo- and heterozygotes at the

Glu298Asp locus between ruptured and unruptured hypertensive patients. This suggests that the profound

effect of hypertension is significant enough that the geneotype is of less importance in determining the

propensity to rupture. Table 6 illustrates that 63% of the hypertensive patients had the optimal genotype

(GG) for Glu298Asp and still went on to rupture, whereas, only 38.2% of the normotensive patients that

ruptured had this preferred geneotype.

Conclusion:

It is unlikely that each of these factors; smoking, hypertension and NOS3 genotype function

independently. The current study has attempted to consider the confounding effect of each factor. It would

be useful in future studies to further consider the interaction of each of these factors in developing any

predictive model. This study is a preliminary effort only and is valuable in establishing that the T-786 C

genotype for NOS3 is not independently associated with any increased risk of rupture when adjusting for

hypertension and smoking. On the contrary Glu298Asp NOS3 genotype is an independent factor associated

with rupture after considering for hypertension and to a lesser extent smoking. This study was ineffective in

addressing the question of increased propensity of aneurysm formation based on the limitations of the control

group.

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In summary, we would suggest that additional studies attempting to provide independent

quantification of the key risk factors associated with aneurysm formation and rupture are needed. Obviously

each risk factor that is studied needs to have a biological or epidemiological basis that can be linked

mechanistically to pathogenesis of aneurysms. These studies will then form the foundation of for a larger

concerted and resource intensive population based prospective study examining the interactive effects of

each of these independent factors and then only can a multifactorial predictive model be developed. It may

prove that the number of factors to be considered is so numerous and that the interactions so complex

making the model illusive. However, we believe that pursuing this will certainly be of more value than our

current efforts that estimate risk of rupture based on a static angiographic image using size as the driving

predictor.

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Table 1. eNOS Genotype Frequencies in Aneurysm Patients and Controls Table 1a Table 1b

eNOS T –786 → C Glu298Asp

Group TT TC CC Total GG GT TT Total Aneurysm (%) 31 (37.8%) 37 (45.1%) 14 (17.1%) 82 53 (55.2%) 34 (35.4%) 9 (9.4%) 96 Controls (%) 73 (39.7%) 87 (47.3%) 24 (13.0%) 184 91 (48.7%) 78 (41.7%) 18 (9.6%) 187

p = 0.69 p = 0.56

Table 2. Baseline Characteristics of the Aneurysm Patients Stratified by Rupture Status.

All Aneurysm Patients

N = 97

Ruptured

Patients

N = 63

Unruptured

Patients

N = 33

Age* 50.7 ± 11.7 48.7 ± 11.2 55.0 ± 11.7

Race (White) 86.6% 87.1% 87.9%

Sex (Female) 79.4% 77.8% 81.8%

Smoker 50.5% 57.1% 50.0%

Hypertensive 44.3% 43.5% 50.0%

Diabetes 2.1% 1.7% 3.1%

Hypercholesterol 4.1% 1.7% 9.4%

CAD 12.4% 10.3% 18.8%

Family History ICH 11.3% 8.6% 18.2%

Age was presented as mean ± SD.

One patient’s aneurysm rupture status was missing.

* p-value = 0.01 for the comparison of ruptured and unruptured patients.

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Table 3. eNOS genotype frequencies in aneurysm patients by rupture status Table 3a Table 3b


Group TT TC CC Total GG GT TT Total Ruptured ICA (%) 16 (34.0%) 22 (46.8%) 9 (19.1%) 47 31 (50.0%) 25 (40.3%) 6 (9.7%) 62

Unruptured ICA (%) 14 (42.4%) 15 (45.5%) 4 (12.1%) 33 22 (68.8%) 8 (25.0%) 2 (6.3%) 32

p = 0.62 p = 0.22

*no adjustment is made for smokers or hypertension Table 4. eNOS genotype frequencies in aneurysm patients stratified by single or multiple aneurysms at presentation. Table 4a Table 4b


Group TT TC CC Total GG GT TT Total 1 aneurysm (%) 12 (32.4%) 19 (51.4%) 6 (16.2%) 37 26 (54.2%) 16 (33.3%) 6 (12.5%) 48

>1 aneurysm (%) 18 (41.9%) 18 (41.9%) 7 (16.2%) 43 26 (56.5%) 18 (39.1%) 2 (4.3%) 46

p = 0.65 p = 0.42

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Table 5. Comparison of eNOS genotype frequencies by rupture status among smokers and non-smokers:

Table 5a: eNOS T –786 → C

Smokers: Non-smokers:

eNOS T –786 → C eNOS T –786 → C Group TT TC, CC Total TT TC, CC Total

Ruptured ICA (%) 9 (34.6%) 17 (65.4%) 26 4 (25.0%) 12 (75.0%) 16 Unruptured ICA (%) 9 (56.3%) 7 (43.8%) 16 5 (31.3%) 11 (68.8%) 16

OR = 4.1, 95%CI = (0.1, 1.5) OR = 0.7, 95%CI = (0.2, 3.5)

Table 5b: Glu298Asp

Smokers: Non-smokers:

Glu298Asp Glu298Asp Group GG GT, TT Total GG GT, TT Total


OR = 0.25, 95%CI = (0.06, 1.04) OR = 0.4, 95%CI = (0.1, 1.5)

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Table 6. Comparison of eNOS genotype frequencies by rupture status among hypertensive aneurysm patients and normotensive aneurysm patients:

Table 6a: eNOS T –786 → C

Hypertensive patients: Normotensive patients:

eNOS T –786 → C eNOS T –786 → C Group TT TC, CC Total TT TC, CC Total

Ruptured ICA (%) 8 (42.1%) 11 (57.9%) 19 7 (25.9%) 20 (74.1%) 27 Unruptured ICA (%) 6 (37.5%) 10 (62.5) 16 8 (50%) 8 (50%) 16

OR=1.21, 95%CI=(0.3, 4.7) OR=0.35, 95%CI=(0.1,1.3)

Hypertension adjusted OR = 0.64, 95%CI = (0.25, 1.61)

Table 6b: Glu298Asp

Hypertensive patients: Normotensive patients:

Glu298Asp Glu298Asp Group GG GT, TT Total GG GT, TT Total


OR=1.02, 95%CI=(0.3, 3.7) OR=0.16, 95%CI=(0.04, 0.65)

Hypertension adjusted OR = 0.42, 95%CI = (0.17, 1.05)

amin kassam, m.d., yue-fang chang, ph.d., robert e. ferrell,...

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