The Gene Encoding Transforming Growth Factor β1 Confers Risk of Ischemic Stroke and Vascular Dementia
Background and Purpose— Transforming growth factor-β1 (TGF-β1) is an anti-inflammatory cytokine that plays an important role in cerebrovascular pathophysiology with protective activity against ischemia-induced neuronal death. We investigated the association of the polymorphism in TGFB1 with ischemic stroke and vascular dementia.
Methods— Three sequence variants in and around promoter and exons of TGFB1 gene were identified in 30 Koreans. Pro10Leu was selected for association study, and then control subjects (n=207) and patients with ischemic stroke (n=271) and vascular dementia (n=207) were screened.
Results— Subjects carrying Leu/Leu were susceptible to both ischemic stroke (odds ratio [OR]=1.63; P<0.05) and vascular dementia (OR=1.88; P<0.01). Analyses with stroke subtypes showed a strong association with small vessel occlusion (SVO, n=110; OR=2.07; P<0.01). Further analysis of SVO data partitioned by gender revealed the female-specific association with Pro10Leu (OR=2.70; P<0.05).
Conclusions— The Pro10Leu of TGFB1 might be a risk factor of ischemic stroke and vascular dementia, especially for SVO in females.
Transforming growth factor-β1 (TGF-β1) is an anti-inflammatory cytokine with neuroprotective activity against ischemia-induced neuronal death,1 and its expression increased in human brain tissue and cerebrospinal fluid after ischemia.2 We investigated the association of the TGFB1 gene with the risk of ischemic stroke and vascular dementia.
Materials and Methods
Patients with ischemic stroke were recruited from Hallym University Hospital with the diagnosis by performing computed tomography or MRI scan from acute stroke patients within 7 days of onset (2002 to 2005). The stoke patients with dementia were excluded based on the criteria described in Kim et al.3 A total of 271 patients with ischemic stroke were further categorized into its subtypes using Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification.4 As a result, 95 patients had large artery atherosclerosis, 110 had small vessel occlusion (SVO), and 20 had cardioembolism. Forty-six cases were diagnosed as stroke with other determined etiology or stroke of undetermined etiology. For details on the patients with vascular dementia and control subjects, see Kim et al.3 Written informed consent was obtained from all subjects. The study protocol was approved by the ethical committee.
Genomic DNA was isolated from peripheral blood cells using a commercially available kit from Qiagen. For detection of sequence variants in TGFB1, promoter, exons 1 to 7, and their flanking regions were screened in 30 random healthy subjects by direct sequencing. For the association study, Pro10Leu polymorphism was genotyped in patients and controls using TaqMan assay. The details on experiments are described in supplemental Appendix I, available online at http://stroke.ahajournals.org.
Pairwise linkage disequilibrium was estimated using SNPAnalyzer (ISTECH Inc). Odds ratios (ORs) and the corresponding CIs were estimated using SAS Release 9.1 (SAS Institute Inc).
Three polymorphisms were discovered at T-509C (promoter), C869T (Pro10Leu, exon1), and G21503A (intron5) by exploring approximately 5.2 kb of TGFB1 (supplemental Appendix II, available online at http://stroke.ahajournals.org). Only Pro10Leu was selected for our association study based on the linkage disequilibrium and allele frequency (supplemental Appendix III, available online at http://stroke.ahajournals.org).
Analyses of clinical characteristics showed no significant difference between disease and control groups in terms of age, gender, hyperlipidemia, and smoking habit (P>0.05; supplemental Table I, available online at http://stroke.ahajournals.org). Hypertension was found in 60.6% of vascular dementia patients and in 58.2% of ischemic stroke patients, which were larger than 32.4% in controls (P<0.05).
Genotypes of Pro10Leu in control and patient groups were in Hardy-Weinberg equilibrium (P>0.05). Because a preliminary result indicated that the distribution of alleles and genotypes did not differ whether subjects had hyperlipidemia, smoking habit, and hypertension (P>0.05; data not shown), these risk factors were excluded in the following association study. The Leu allele increased the risk of vascular dementia (OR=1.53; P<0.01; Table 1), and subjects carrying its homozygous genotype were more susceptible to both ischemic stroke (OR=1.63; P<0.05) and vascular dementia (OR=1.88; P<0.01). In analyzing the subtypes of ischemic stroke, an association of the genotypes with SVO was observed in a recessive model (OR=2.07; P<0.01; Table 1). The association remained significant when multiple comparison tests were used with Bonferroni correction (P= 0.018; supplemental Table II, available online at http://stroke. ahajournals.org).
Because a preliminary result showed not only gender effects but also their interaction effects with the genotype (P<0.05; data not shown), we further analyzed the data partitioned by gender. Leu/Leu was significantly associated with ischemic stroke (OR=2.06; P<0.05) and vascular dementia (OR=2.12; P<0.05) in females, but not in males (P>0.05; Table 2). This gender-specific effect was also present for SVO. The OR estimate in the recessive model for female SVO patients was 2.70, and its significance increased even with the small sample size (P=0.014). It was significant even after Bonferroni correction (P=0.042; supplemental Table II).
Ischemic stroke is a clinically heterogeneous disorder encompassing strokes attributable to various etiologies, and thus we investigated heterogeneous genetic effects of TGFB1 on the subtypes. The effect of Leu/Leu was confined to SVO among the stroke subtypes. Although the pathogenesis of SVO is not clearly understood, thrombosis is a great contributor to SVO. TGF-β1 is an anti-inflammatory cytokine that is believed to play a key role in thrombosis. Therefore, TGFB1 might be related to the thrombotic occlusion of small vessels by regulating inflammatory process.
Further analysis of data partitioned by gender revealed that Leu/Leu was susceptible to SVO in females, but not in males. In complex traits, gender-specific effects of autosomal genes can be usually explained by epistasis with sex-linked genes or by interaction with nongenetic factors that are correlated with sex. The epistasis with sex-linked genes often results from hormonal effects on gene expression and regulation. The female-specific effect found in this study could be explained by TGF-β1 dependent on estrogen level. The genotype Leu/Leu was associated with decreased TGF-β1 concentration.5 Cross-regulation of TGF-β1 and estrogen has been reported by showing increased mRNA expression from estrogen treatment and increased transcriptional activity of estrogen receptor from activation of the TGF-β1 signaling pathway.6 Postmenopausal estrogen use decreased the risk of stroke in human.7 In conclusion, the low levels of TGF-β1 in females with the Leu/Leu genotype could make the expression of estrogen receptor decrease, which might eventually lead to the corresponding decline in the protective function of estrogen on stroke.
A stronger impact on vascular dementia than on ischemic stroke might imply the additional influence of TGFB1 on the pathogenesis related to cognitive impairment or memory function after stroke event in patients with vascular dementia. Further study with subtypes of vascular dementia would provide more clear explanation of the additional impact on the subtypes.
In conclusion, the current study suggested that the Pro10Leu in TGFB1 might be a risk factor for SVO, particularly in females. Enormous association studies for the subtypes with other genes should precede to elucidate their genetic architectures.
Supplemental Appendix I
Genomic DNA was isolated from peripheral blood cells using QIAamp DNA blood mini kit (Qiagen). For detection of sequence variants in TGFB1 gene, promoter region, exons 1 to 7, and their flanking regions were amplified by PCR, and then sequenced using the ABI PRISM 3730 DNA analyzer (Applied Biosystems) in 30 random healthy subjects. All reactions were carried out according to the manufacturer’s protocol. Primers were designed based on GenBank sequences (supplemental Table III, available online at http://stroke.ahajournals.org).
Pro10Leu polymorphism for the association study was genotyped in 271 ischemic stroke patients, 207 vascular dementia patients, and 207 controls using the TaqMan PCR assay (Applied Biosystems). After the reactions following the manufacturer’s protocol, the products were analyzed using ABI PRISM 7900HT (Applied Biosystems). The sequences of the primers and probes for Taqman assay are summarized in supplemental Table IV, available online at http://stroke.ahajournals.org. Genotyping was performed by laboratory personnels blind to case-control status of the samples. For validation, 10% randomly selected from the genotyped samples were confirmed by direct sequencing, and the concordance rate for quality control samples was 100%.
Supplemental Appendix II
Discovery of SNPs in TGFB1
Approximately 5.2 kb of TGFB1 gene were explored to discover sequence variants by direct sequencing. Three polymorphisms from 30 subjects were discovered at T-509C in the promoter region, at C869T in exon 1, and at G21503A in intron 5 (supplemental Figure I, available online at http://stroke.ahajournals.org). The transition of C→T at the polymorphism of C869T led to amino acid substitution from proline to leucine at codon 10 in exon 1, and it was referred as Pro10Leu. The segregations at positions −988, −800, codon 25, and codon 263 reported from other ethnic groups1–3 were not observed in our population.
Supplemental Appendix III
SNP Selection for Association Study
Of the 3 polymorphisms, only Pro10Leu polymorphism was examined for our association study. The G21503A was excluded because of its rare allele frequency (1.7%, supplemental Table V, available online at http://stroke.ahajournals.org). The estimates of linkage disequilibrium (|D′|=1.00; r2=0.94) revealed an almost complete linkage between T-509C and Pro10Leu (supplemental Table V), which concurred with the findings from Cambien et al1 (|D′|=0.95) and Kim et al4 (|D′|=0.99), and the analysis of both loci was redundant. The selection of Pro10Leu was attributable to the presence of its influence on the secretion of TGF-β1.5 On the other hand, T-509C was not located in any known consensus sequence for a promoter regulatory element, and its role in transcription was thought to be doubtful.
Source of Funding
This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A020007).
- Received July 10, 2006.
- Revision received July 22, 2006.
- Accepted August 17, 2006.
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