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(Stroke. 2003;34:2817.)
© 2003 American Heart Association, Inc.
Original Contributions |
From the Departments of Neurosurgery (D.K., G.S.) and Surgical Research (H.K.S.), University Hospital Carl Gustav Carus, University of Technology, Dresden, and Institute of Medical Biometry and Statistics (A.Z., I.R.K.), University of Lübeck, Lübeck, Germany.
Correspondence Dr Dietmar Krex, Department of Neurosurgery, Carl Gustav Carus Hospital, University of Technology, Fetscherstr 74, 01307 Dresden, Germany. E-mail krex{at}rcs.urz.tu-dresden.de
| Abstract |
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Methods A study sample was analyzed that comprised 44 patients with intracranial aneurysms and 44, 41, and 40 controls for the analysis of TIMP-1, -2, and -3, respectively. Differences in genotype and allele frequencies of identified polymorphisms were determined. The entire coding regions and parts of the promoter sequences of the TIMP-1, -2, and -3 genes were with using the automated laser fluorescence technique.
Results Nine polymorphisms were identified, 3 located in TIMP-1 (-19C>T, 261C>T, 372T>C), 4 in TIMP-2 (-621C>T, -596A>C, -261G>A, 303G>A), and 2 in TIMP-3 (249T>C, 261C>T), whereas -621C>T, -596A>C, and -261G>A of the TIMP-2 gene are newly identified polymorphisms. We detected no deviation from Hardy-Weinberg equilibrium in any of the groups. The C allele of the 372T>C polymorphism was more frequently found in female than in male controls (exact nominal P=0.0012). However, this finding could not be validated by analysis of a second sample of 113 controls (exact nominal P=1.0000). There were no differences in genotype and allele frequencies between any of the other groups.
Conclusions Our analysis of the entire coding region of 3 TIMPs, which are the main inhibitors of metalloproteinase activity in the extracellular matrix, failed to show an association between genetic polymorphisms and an intracranial aneurysm. These data do not support the hypothesis that genetic variants within these genes have an impact on aneurysm development in the white population.
Key Words: connective tissue intracranial aneurysm polymorphism (genetics) risk factors
| Introduction |
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There is evidence that remodeling of the extracellular matrix (ECM) plays a major role in the pathogenesis of cerebral aneurysms. The ECM is a dynamic network of proteins and proteoglycans that not only is essential for structural maintenance in many tissues but also is involved in cell adhesion and migration, as well as in cell proliferation and differentiation processes. The structural integrity of the vessel wall relies on a balance between synthesis and degradation of ECM proteins. However, increased circulating levels of proteases, which have been detected in some aneurysm patients,5 diminished arterial structural proteins,6 exaggerated expression of catalytic enzymes in the aneurysm wall,7 and a predominance of superoxide anion metabolites8 all point to an imbalance in favor of protein degrading processes in the arterial wall. Remodeling of the vascular ECM has been found to be involved in the pathogenesis of other vascular diseases like aortic aneurysm,9,10 atherosclerosis,11 and postangioplasty restenosis.12,13 Among the degrading enzymes, the growing family of matrix metalloproteinases (MMPs) has been found to be crucial in ECM remodeling of vascular walls. An important mechanism for the regulation of the activity of MMPs is equimolar binding to a family of homologous proteins referred to as tissue inhibitors of metalloproteinases (TIMP-1 through TIMP-4) (a review is given by Brew et al14). TIMP-3, unlike the other 3 TIMPs, binds tenaciously to ECM components. Mutations in the TIMP-3 gene have been found in patients with Sorsbys fundus dystrophy, an autosomal dominant inherited macular disorder, characterized by accumulation of abnormal deposits in the Bruchs membrane and choroidal neovascularization.15 The most recently isolated TIMP-4 also binds to pro-MMP-2; however, so far there are no data about its clinical significance.16,17
The increased proteolytic activity observed in the vascular wall of aneurysm patients may be due to either an elevated activity of proteolytic enzymes or diminished activity of their inhibitors. Genetic variants of the encoding genes might result in altered enzyme activities of each protein.
We conducted this case-control study to investigate whether polymorphisms in the promoter and coding regions of the TIMP-1, -2, and -3 genes are associated with the occurrence of cerebral aneurysms in white populations.
| Subjects and Methods |
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The control groups consisted of 44, 41, and 40 anonymous, healthy blood donors enrolled from the same urban area for the analysis of TIMP-1, TIMP-2, and TIMP-3, respectively. Differences in sample size were due to difficulties in generating a complete sequence analysis of all 3 genes for all 44 controls. We must emphasize that the control group was enrolled from the same geographic area as the cases. Because of its historical background, Dresden is populated by a homogenous white population. In general, blood is donated by all social classes in Germany. All potential blood donors are routinely screened for disease with a questionnaire, red blood cell count, white blood cell count, and liver function parameters (details provided on request). A second control sample was recruited from the same population of blood donors and comprised another 113 (43 men, 70 women) controls for the analysis of TIMP-1. This study was approved by the local ethics committee. Informed written consent for genetic analysis was obtained from all nonanonymous individuals.
Sequence Analysis
For amplification of genomic DNA, primer pairs were designed on the basis of the genomic sequence of the TIMP genes published in GenBank (Accession NT_011584, U44381-85, NT_0115200). Primers are located in the adjacent intronic sequences of each exon, allowing analysis of exon/intron borders. In addition, 483, 676, and 418 bp of the TIMP-1, -2, and -3 promoter regions were analyzed, respectively.
Polymerase chain reaction (PCR) was performed with a thermal cycler (Perkin Elmer, Applied Biosystems GmbH). For negative control, water was used instead of genomic DNA in PCR.
PCR products were electrophoresed on an 0.8% agarose gel, and bands were cut out and eluted. Three microliters of the eluted PCR product were used in the reaction mix to which 1 µL of Thermo Sequenase Mix (Amersham Pharmacia Biotech, UK Ltd) and 1 µmol/L cyanine5 end-labeled sense primer were added. The cycling reaction also was performed with a thermocycler. Cycling products were run on A.L.F.express with denaturing 6.5% Long Ranger gels, which were run at 40 W (1000 V, 38 mA) for 4 hour. Runs were analyzed with A.L.F. Evaluation software. The primer list and details on PCR and cycling reaction conditions can be provided on request.
Analysis of the second study population for TIMP-1 polymorphisms was performed with mutation detection by melting point analysis on a real-time PCR device (LightCycler). Primers and hybridization probes were designed covering the 372T>C polymorphism. Three samples with known genotypes and a negative control were used in each run. Primer sequences and PCR conditions will be provided on request.
Study Course and Statistical Methods
For all variants investigated, deviations from Hardy-Weinberg equilibrium were evaluated by comparing observed and expected genotype frequencies by an exact goodness-of-fit test separately in cases and controls. Odds ratios (ORs) and exact 95% confidence intervals (CIs) were calculated to compare allele frequencies18 and genotype frequencies using the Cochrane-Armitage trend test.
Nomenclature
The location of the polymorphisms is given according to the nomenclature system recommended by Antonarakis19 and the Nomenclature Working Group 1998 (http://www.dmd.nl/mutnomen.html).
| Results |
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In TIMP-2, we identified 4 polymorphisms. Three of those-261G>A, -596A>C, and -621C>Tare located in the promoter region and have not been described before. Another, 303G>A, is located in exon 3 without having an effect on the amino acid sequence (S101S). Genotype and allele frequencies of these polymorphisms are presented in Tables 3 and 4
, together with ORs and exact 95% CIs. No deviation from Hardy-Weinberg equilibrium was observed in either cases or controls (all nominal P>0.05). Allele and genotype frequencies between cases and controls did not differ (all nominal P>0.05).
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TIMP-3 harbored 2 polymorphisms, 249T>C and 261C>T, both located in exon 3, and neither changes the amino acid sequence (H83H and S87S, respectively). The genotype and allele frequencies are presented in Tables 5 and 6
, together with ORs and exact 95% CIs. Again, no deviation from Hardy Weinberg equilibrium and no difference between cases and controls in allele or genotype frequencies were observed (all nominal P>0.05).
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| Discussion |
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To investigate the possible impact of gene polymorphism on the development of intracranial aneurysms, several candidate genes have been analyzed in different ethnic groups.2026 Enzymes of the ECM are a reasonable target, as elevated activity and /or overexpression of pro-MMP-2, MMP-2, MMP-9, and MT1-MMP in the serum of patients with cerebral aneurysms and in the aneurysm wall have been reported.5,7,27 MMPs are the principal matrix-degrading proteinases.28 The expression of most MMPs is transcriptionally regulated by various factors, whereas their activity is additionally regulated by zymogen activation and controlled by endogenous inhibitors in the serum and tissue. TIMPs are considered to be the key inhibitors of MMPs in the ECM. The functional importance of TIMPs in the pathogenesis of aneurysms has been demonstrated by Allaire et al29 using a rat model for local overexpression of TIMP-1. This resulted in the maintenance of structural integrity and prevented aneurysm development. The hypothesis that TIMPs might play a role in cerebral aneurysm pathogenesis has been supported by recent findings from Peters et al,30 who have reported a global gene expression analysis of a single intracranial aneurysm from a 3-year-old girl. Among the 25 most significant tags overexpressed in intracranial aneurysms, they identified TIMP-3. A genetic variant in that particular gene might be the cause of its overexpression. However, in our study, we failed to show such an association, which might be explained by the following reasons. The investigation by Peters et al is based on the analysis of a single case, a 3-year-old girl, presenting with multiple aneurysms of the middle cerebral artery, and no other clinical data are given. The incidence of cerebral aneurysms in childhood is
1% to 2% of all aneurysms.31 In contrast, the mean age in our study population was 52 years, which represents the decade in which most of the aneurysms are found in men, and the peak incidence in women is at an even older age.32,33 The genesis of an aneurysm is a multifactor event. Hypertension, arteriosclerosis, and long-lasting elevated shear stress are widely accepted risk factors in the pathogenesis of spontaneous intracranial aneurysms (for a review, see Krex et al34); however, these are not found in childhood. Hence, it might be argued that multiple aneurysms in early childhood are based on a pathogenesis different from that commonly found in spontaneous aneurysms of adults, eg, an inherited connective tissue disease or polycystic kidney disease. Still, our results do not exclude the possibility that TIMP-3 is involved in the pathogenesis of aneurysms.
We analyzed the first 304 bp of the TIMP-3 promoter and the first 114 bp of the 5'UTR. The first 112 bp of the promoter harbors multiple Sp1 binding sites, and a potential NF I site is located between -235 and -248.35 This region has been shown to be elementary for TIMP-3 basal activity and serum inducibility.35 Nevertheless, we analyzed only a part of the entire promoter region and did not screen for posttranslational modifications or epigenetic effects that might influence the expression of a gene.
TIMP-2 is an important inhibitor of MMP-2 but also of other active MMPs.36 Butler et al37 have shown that the specificity of TIMP-2 for MMP binding and inhibition can be modified by single amino acid mutations. These results support the hypothesis that genetic variants might be the cause for altered enzyme activities in the ECM of aneurysm walls, although our results did not substantiate this.
Wang et al38 have analyzed the sequences of the TIMP-1 and TIMP-2 genes in a mixed North American and European white population with abdominal aortic aneurysm, but also in 1 individual with both aortic and intracranial aneurysms and 4 individuals with intracranial aneurysms. They identified 2 polymorphisms each in TIMP-1 and TIMP-2 and presented preliminary results of different allele frequencies of a nucleotide 573 polymorphism in the TIMP-2 gene between male controls and patients with abdominal aortic aneurysms. However, they failed to show a significant association with the phenotype, especially with cerebral aneurysms. In our study, we detected the identical polymorphisms, although we refer to a different nomenclature for the polymorphisms, and the findings of Wang et al in another ethnic group are in accordance with our results.
In conclusion, although there is growing evidence that enzymes of the ECM play a major role in the pathogenesis of intracranial aneurysms, our results failed to show that genetic variants within the coding regions of TIMP-1, -2, and -3, which might influence the biochemical properties of the enzyme, have an impact on cerebral aneurysm development. However, expression of a gene might be regulated by sequences located several kilobases upstream from the transcription start site, as shown by Dean et al39 for the TIMP-1 gene, suggesting that analysis of a long promoter sequence is mandatory.
| Acknowledgments |
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Received March 10, 2003; revision received June 11, 2003; accepted August 1, 2003.
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