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the Departments of Neurology (A. Slowik, A.B., W.T., J.P., A.T.D., A. Szczudlik) and Neurosurgery (M.B., T.K., R.C.), Jagiellonian University, Krakow, Poland
the Department of Neurology (D.A.F.), University of Michigan, Ann Arbor.
Abstract
Background and Purpose— The member 3 of clade A of serine proteinase inhibitors (SERPINA3), known previously as the 1-antichymotrypsin, is an acute phase protein, the levels of which increase in acute and chronic inflammation. The A/T polymorphism of the SERPINA3 gene influences expression of SERPINA3 protein. SERPINA3 can be related to aneurysmal subarachnoid hemorrhage (SAH) by influencing inflammation or by regulating cathepsin G activity. We studied the significance of SERPINA3 A/T polymorphism in patients with aneurysmal SAH compared with healthy controls.
Methods— A total of 180 patients with aneurysmal SAH and 263 healthy controls were genotyped for the SERPINA3 A/T polymorphism. Aneurysmal SAH was diagnosed by cranial computed tomography or lumbar puncture and digital subtraction angiography. SERPINA3 polymorphism was detected by polymerase chain reaction amplification and restriction enzyme digestion.
Results— The SERPINA3 genotype distribution in patients with aneurysmal SAH (AA-29 16.1%; AT-108 60.0%; TT-43 23.9%) differed significantly from controls (AA-70 26.6%; AT-123 46.8%; TT-70 26.6%; P=0.009). A logistic regression model showed that the presence of genotype with T allele (AT+TT; odds ratio [OR], 2.01; 95% CI, 1.19 to 3.38; P=0.009) or AA genotype (OR, 0.49; 95% CI, 0.30 to 0.84; P=0.009) of the SERPINA3 influences the risk for aneurysmal SAH independently from smoking, excessive alcohol consumption, and hypertension.
Conclusion— The A/T polymorphism of SERPINA3 gene is associated with the risk factor for aneurysmal SAH.
Key Words: alpha 1-antichymotrypsin polymorphism SERPINA3 subarachnoid hemorrhage
Introduction
The member 3 of clade A of serine proteinase inhibitors (SERPINA3) is an acute phase protein produced in the liver, the concentration of which can rise during acute1 and chronic inflammation.2 SERPINA3 is also an inhibitor of cathepsin G, which is involved in degradation of the extracellular matrix proteins.1
SERPINA3 gene is located on chromosome 14q32.1. The A/T polymorphism changes amino acid alanine to threonine at codon 15 in the signal peptide region.3 Experimental4 and clinical studies postulate that SERPINA3 A/T polymorphism influences the expression and plasma levels of SERPINA3 protein.2,4 It is not known whether this polymorphism affects the inhibitory function of SERPINA3.
The concept of genetic susceptibility for subarachnoid hemorrhage (SAH) from ruptured aneurysm is increasingly supported. Studies have shown that the family history of aneurysmal SAH5,6 and some polymorphisms of genes involved in tissue remodeling, such as II genotype of angiotensin-converting enzyme gene,7 significantly increase risk for the disease. Japanese and Finnish studies identified in DNA linkage studies 2 chromosomal regions, located on chromosome 7q11 and 19q13.3, respectively, as loci that predispose to intracranial aneurysms.8,9
Data suggest that inflammation10–12 and extracellular matrix remodeling13 play a role in aneurysmal SAH. SERPINA3 can be potentially involved in the pathogenesis of aneurysmal SAH by influencing these 2 processes. We conducted the study to determine whether the A/T polymorphism of SERPINA3 gene influences the risk for aneurysmal SAH in a Polish population.
Materials and Methods
Study Population
We included 180 unrelated patients with a diagnosis of SAH from ruptured saccular aneurysm out of a total of 323 patients with SAH admitted to the Stroke Unit and Neurosurgery Department at Jagiellonian University, Poland, between 2002 and 2004. Patients with dissecting and fusiform aneurysms (n=13), arteriovenous malformations (n=25), or unknown origin of SAH (n=32), or who were comatose at admission (n=41) or were not agreeing to participate in this study (n=32) were excluded. The dropout of patients with aneurysmal SAH was 28.85%.
In each case at admission, we performed cranial computed tomography or lumbar puncture to confirm the SAH diagnosis. The diagnosis of intracranial saccular aneurysms was established by digital subtraction angiography. We also included 263 unrelated controls, matched for age (±2 years) and sex with patients, free of clinically detectable cerebrovascular disease and without any stroke history. They were recruited from the beginning of the study. Thirty percent of them were the spouses of the patients admitted to our stroke unit. They were not necessarily the spouses of the patients enrolled in the study. The spouses were not matched with their relatives affected with aneurysmal SAH but with other subjects of the same age and sex. All subjects were white and came from a province in southern Poland. Before inclusion in the study, all participants gave informed consent. The study was approved by the university ethical committee and was performed in accordance with the Helsinki Declaration of 1975, as revised in 1983.
For all subjects from each study group, we collected demographic data and risk factor profile. Patients and controls received a detailed clinical questionnaire including information on vascular risk factors, current medication, and physical examination. Answers were verified by analysis of the medical documentation, and for SAH patients, by the information from their proxies. The individual was classified as having arterial hypertension if he/she met 1 of the following criteria: (1) diagnosis of hypertension in previous medical history; (2) antihypertensive treatment before entry to the study; or (3) systolic or diastolic blood pressure 140 mm Hg or 90 mm Hg, respectively, on at least 2 different occasions (the first 3 days of hospitalization were not considered for the SAH patients).
History of ischemic heart disease was established on past medical history, examination of previous and current electrocardiograms and laboratory data.
Smoking habits were defined as current smokers of 1 cigarette per day, former smokers, or nonsmokers. For statistical analysis, "current smokers" and "former smokers" were pooled together. Excessive alcohol intake was defined as alcohol consumption of >300 g per week (>3 alcoholic drinks daily).
Laboratory Techniques
Genetic analyses were performed by laboratory personnel who were kept blind to sample identity. Leukocyte DNA was extracted using a commercially available kit (High Pure PCR Template Preparation Kit; Roche Molecular Biochemicals).
Genotyping for the SERPINA3 A/T polymorphism was performed using polymerase chain reaction (PCR) and restriction enzyme digestion. The PCR was performed based on Vila et al.15
The reaction was performed in a final volume of 15 μL containing 7 pmol/L of each primer, 10X buffer (Finnzyme), 0.2 mmol/L of 2'-deoxynucleoside 5'-triphosphate (Sigma), 0.4 U of Taq DNA polymerase (Finnzyme), and 150 ng of human genomic DNA to yield a 124-bp DNA product.
The sequence of the sense primer was 5' CAG AGT TGA GAA TGG AGA 3', and the reverse primer was 5' TTC TCC TGG GTC AGA TTC 3'. PCR was performed using the Uno apparatus (Biometra) for initial denaturation at 94°C for 7 minutes and for 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 45 seconds and the final extension step at 72°C for 7 minutes.
The 124-bp product was then digested with Mva I (Promega). Digestion was performed in a 18-μL reaction with 15 μL of PCR product, 10X buffer Y+ with BSA (MBI Fermentas), and enzyme. Reaction was incubated at 37°C for 12 hours. The resulting fragments were then separated in a 3.5% ethidium bromide–stained agarose gel. In TT homozygotes, the restriction digest resulted in a 117-bp fragment; in AA homozygotes, 2 bands of 84 bp and 33 bp; and in AT heterozygotes, 3 bands of 117 bp, 84 bp, and 33 bp. The first 100 samples from the study subjects were run twice independently. The concordance rate of reading them by the 2 independent readers was equal to 100%. Then in the rest of cases, we performed the DNA analysis once. These results were interpreted by 2 independent persons. In case of different readings (3%), the samples were run 1 more time and still read by 2 or 3 independent persons.
Statistical Analysis
Data on continuous characteristics are expressed as means±SD. Data on categorical characteristics are expressed as percent values or absolute numbers as indicated. Significance level for genotypes distribution (3x2 table) was calculated using Fisher exact test and 2 tests.
Comparisons between groups were made with 2 test (nominal data) or Student’s t test (interval data). A value of P<0.05 was considered statistically significant. Hardy–Weinberg equilibrium was tested by the 2 method. The power of our study assessed by the software calculating the power for genetic association studies16 equals 21.75%. The association of the SERPINA3 A/T genotype with the risk for aneurysmal SAH was investigated by means of logistic regression analysis, considering potential confounding risk variables including age, sex, and other conventional risk factors. For multivariate risk predictors, the adjusted odds ratios (ORs) are given with the 95% CIs. Calculations were performed using the commercial statistical package STATISTICA for Windows, version 6.0 (StatSoft, Inc).
Results
We found 241 aneurysms in 180 patients with aneurysmal SAH. Their locations were as follows: anterior cerebral artery complex 73 (30.3%); middle cerebral artery complex 84 (34.9%); internal carotid artery complex 64 (26.5%); and vertebrobasilar artery complex 20 (8.3%). Characteristics of patients with aneurysmal SAH and their controls are shown in Table 1. Patients with aneurysmal SAH presented significantly more often with hypertension, current or previous smoking, and excessive alcohol intake.
SERPINA3 genotype and allele frequencies are presented in Table 2. Genotype frequency in the control group was in Hardy–Weinberg equilibrium. Patients with aneurysmal SAH deviated from Hardy–Weinberg equilibrium (P<0.01). Significance level for genotype distribution (3x2 table) calculated using Fisher exact or 2 tests was equal to 0.01.
We found that genotypes with T allele compared with AA genotypes are present more frequently in patients with aneurysmal SAH than in the controls (OR, 1.89; 95% CI, 1.16 to 3.06; P=0.009). There is also a statistically significant difference between the distribution of AT genotypes compared with AA genotypes in the 2 studied groups (OR, 2.12; 95% CI, 1.28 to 3.52; P=0.004). We have not found any difference between the groups when we compare the frequencies of TT genotypes with AA genotypes (OR, 1.22; 95% CI, 0.91 to 1.63; P=0.18), TT genotypes with genotypes with A allele (OR, 1.27; 95% CI, 0.70 to 2.30; P=0.42), or TT genotypes with AT genotypes (OR, 0.73; 95% CI, 0.44 to 1.21; P=0.21). ORs are unadjusted for risk factor and demographics. There is no significant difference in A versus T allele distribution between the studied groups.
The distribution of SERPINA3 genotype and allele frequencies was similar in patients with single (n=138) and multiple aneurysms (n=42): AA 20 (14.5%), AT 83 (60.1%), and TT 35 (25.4%) versus AA 9 (21.4%), AT 25 (59.5%), and TT 8 (19.1%), respectively (P=0.3); and A 123 (43.0%) and T 153 (57.0%) versus A 43 (48.9%) and T 41 (51.1%), respectively (P=0.2).
After adjustment for the studied risk factors, logistic regression analysis has shown that the genotypes with T allele (OR, 2.01; 95% CI, 1.19 to 3.38; P=0.009) or AA genotype of SERPINA3 (OR, 0.49; 95% CI, 0.30 to 0.84; P=0.009), hypertension (OR, 2.47; 95% CI, 1.58 to 3.87; P=0.00007), smoking (OR, 2.94; 95% CI, 1.92 to 4.53; P=0.000001), and excessive alcohol intake (OR, 2.49; 95% CI, 1.15 to 5.24; P=0.02) independently affect the risk for aneurysmal SAH in the studied group of patients.
Discussion
This study has shown that genotypes with T allele increases the risk for aneurysmal SAH; however, the alternative interpretation could be that AA genotype of SERPINA3 decreases the risk for this disease.
Although the only statistically significant difference between the frequency of the genotypes was found when we compared AA genotypes with AT genotypes, we decided to combine genotypes with T allele together (AT and TT) and we compared them with AA genotypes on the basis of the results from studies suggesting that T allele influences significantly higher expression of SERPINA3 than A allele. Unfortunately, available data on this topic are scarce and indirect. It has been shown that cells transfected with G or T alleles of the promoter region of SERPINA3 G/T polymorphism, being in complete linkage disequilibrium with a signal peptide A/T polymorphism, display different expression of SERPINA3.4 The effect of G/T polymorphism on luciferase reporter gene activity in Hep G2 and T98G cells shows that both basal and stimulated by oncostatin M expression of SERPINA3 in cells transfected with T allele is significantly higher compared with cells transfected with G allele.
There are also clinical studies showing that SERPINA3 A/T polymorphism affects SERPINA3 plasma levels in chronic inflammatory condition, with the highest levels in TT carriers compared with AA carriers.2,4 However, some studies have not shown a relationship between the polymorphism and levels of SERPINA3.17 These discrepancies may be attributable to the fact that SERPINA3 levels are highly variable and sensitive to different peripheral inflammatory conditions, which may influence the function of SERPINA3 AA, AT, and TT proteins.18 Because several other polymorphisms of SERPINA3 gene have been found (ie, promoter G/T, codon 76, codon 227, codon 241, codon 250, codon 324, intron 4, and 3'untranslated region), it is also possible that the nonrandom combination of risk and protective alleles may influence the clinical significance of SERPINA3 signal region A/T polymorphism.19
The possible link between aneurysmal SAH and SERPINA3 could be the presence of local inflammation in the wall of ruptured aneurysm. The ruptured aneurysmal wall presents with increased expression on protein level of several proinflammatory molecules,11 as well as increased mRNA levels of major histocompatibility complex class I and II markers and IgG heavy chain and Ig light chain.12 The endothelium of ruptured aneurysms is invaded with macrophages and leukocytes.10 The inflammatory cells secrete different proteases, including cathepsin G.
Cathepsin G destroys the extracellular matrix proteins,14 making the vessel wall more fragile, and induces angiotensin II–generating system that exerts potent local vasoactive and chemoattractant properties at sites of inflammation.20 SERPINA3 inhibits specifically neutrophil cathepsin G.21 Local expression of SERPINA3, dependent on its polymorphism, may control the inflammatory response and vascular remodeling. It has been shown previously that aneurysmal SAH is the disease with the decreased activity of renin-angiotensin system.22
It cannot be excluded that systemic chronic inflammation accompanies aneurysmal SAH. The implication of SERPINA3 protein as a marker of systemic inflammation preceding the disease comes only from the studies on Alzheimer disease.23,24 The Rotterdam Study showed that the levels of SERPINA3 and other inflammatory proteins were increased many years before the onset of Alzheimer disease and vascular dementia.24 At present, no study has examined whether chronic systemic inflammation precedes aneurysmal SAH; there are no data assessing the relationship between the SERPINA3 levels or levels of other inflammatory proteins and the risk for this disease. Interestingly, there are data showing that some markers of inflammation (eg, C-reactive protein) increase risk for subsequent ischemic stroke,25 so it is certainly possible that a similar process may pre-exist SAH. The best way to answer whether SERPINA3 A/T polymorphism influences the risk for subsequent aneurysmal SAH would be a population-based long-term follow-up study, with serial assessments of SERPINA3 levels. However, this design would require studying a very large population.26
The significance of several conventional risk factors, such as hypertension,5 heavy alcohol consumption,27 and smoking,5,27 has also been identified so far, and their roles were confirmed in our study. The magnitude of risk associated with the presence of genotype with T allele or AA genotype of SERPINA3 gene is lower than for above-mentioned risk factors. This may be attributable to the lower impact of that risk factor on overall incidence of SAH or to the smaller power of our study when compared with systematic reviews28 or both.
Unfortunately, we were able to obtain family history for aneurysmal SAH from only 65% of our SAH patients. The family history for aneurysmal SAH was reported by 3 of 117 patients (3.5%) and by 2 out 263 of controls (0.8%), and the difference was not statistically significant (P=0.15). Because this is a proven risk factor for this disease, the lack of the information from all cases could, to some degree, influence the independent predictors for aneurysmal SAH in a logistic regression model.
The spouses of the patients were included into the control group, and this could be responsible for the "healthy worker effect." It is possible that this subgroup of controls more likely participates in studies and therefore less likely has a gene that would predispose to multiple medical problems. This encouraged us to compare the genotype frequencies of SERPINA3 A/T polymorphism in our controls with the frequency of the genotypes in different controls of white origin.17,19 We have found no significant differences between the groups.
We are aware that up to 5% of our controls may have an unruptured aneurysm. We can expect 13 cases with unruptured aneurysms of the total 263 controls. It is very likely that it does not influence the final results, or it may cause an underestimate of the true OR.
The shortcoming of our study is the lack of assessment of the SERPINA3 levels, which could answer the following questions: (1) are SERPINA3 levels higher in patients with aneurysmal SAH compared with controls and (2) are SERPINA3 levels influenced by SERPINA3 polymorphism in patients with aneurysmal SAH It would also be interesting to correlate the SERPINA3 levels with other measures of inflammation, such as C-reactive protein, that were not assessed in our study.
Our study indicates that SERPINA3 A/T polymorphism is a risk factor for aneurysmal SAH. Future studies should answer the question of which genotype of this polymorphism plays a role in the risk for aneurysmal SAH.
Acknowledgments
This research was supported by State Committee for Scientific Research grant 6PO5B 049 20 and the Research Grant from the Jagiellonian University 501/KL/634/L. We thank Anna Dziubek, Halina Kaliszuk, Malgorzata Sado, and Agnieszka Kieltyka for their invaluable technical assistance, and Dorota Wloch for administrative support.
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