Journal of the Neurological Sciences 341 (2014) 55–57
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Genetic variations of MMP9 gene and intracerebral hemorrhage susceptibility: A case–control study in Chinese Han population Jie Yang a,b,1, Bo Wu a,1, Sen Lin a, Junshan Zhou b, Yingbin Li c, Wei Dong a, Hisatomi Arima d, Chanfei Zhang a, Yukai Liu b, Ming Liu a,⁎, for the Chengdu stroke registry and Nanjing First Hospital stroke registry investigators a
Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, China Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Neurosurgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China d The George Institute for Global Health, Royal Prince Alfred Hospital and University of Sydney, Sydney, NSW 2050, Australia b c
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 23 March 2014 Accepted 25 March 2014 Available online 2 April 2014 Keywords: Matrix metalloproteinase 9 Single nucleotide polymorphism Genotype Haplotype Intracerebral hemorrhage Etiology
a b s t r a c t Objective: To investigate the association between genetic variations of matrix metalloproteinase 9 (MMP9) gene and intracerebral hemorrhage (ICH) susceptibility in Chinese Han population. Methods: The clinical data and peripheral blood samples from the patients with ICH and hypertension, and controlled subjects with hypertension only, were collected. MassARRAY Analyzer was used to genotype the tagger single nucleotide polymorphism (SNP) of MMP9 gene. Haploview4.2 and Unphased3.1.7 were employed to construct haplotypes and to analyze the association between genetic variations (alleles, genotypes and haplotypes) of MMP9 gene and ICH susceptibility. Results: 181 patients with ICH and hypertension, and 197 patients with hypertension only, were recruited between Sep 2009 and Oct 2010. Patients in the ICH group were younger (61.80 ± 13.27 vs. 72.44 ± 12.71 years, p b 0.05). Other conventional risk factors between the ICH and control groups were similar. There were 6 Tagger SNPs and 4 haplotypes of MMP9 gene in our sample population. Our logistical regression analysis showed that there were no significant associations between genetic variations of the MPP9 gene and ICH susceptibility (all p N 0.05). Conclusions: The genetic variations of MMP9 gene were not significantly associated with ICH susceptibility in the Chinese Han population. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute spontaneous intracerebral hemorrhage (ICH) is estimated to occur in ≈5 million people worldwide each year, and most of whom either die or are left seriously disabled because of limited effective treatment strategies available [1–3]. Prevention of ICH requires better knowledge on genetic susceptibility as well as on conventional risk factors. Most primary ICH originates from bleeding at small arteries, which are characterized by breakage of extracellular matrix in arterial walls [4]. Matrix metalloproteinase 9 (MMP9) can damage small vessels through degrading the extracellular matrix and may cause ICH [5,6]. Increased plasma MMP9 levels were also observed in patients with
⁎ Corresponding author at: Stroke Clinical Research Unit, Department of Neurology, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Chengdu 610041, China. Tel.: +86 28 8181 2671; fax: +86 28 8542 3551. E-mail address: [email protected] (M. Liu). 1 The first two authors should be regarded as co-first authors.
http://dx.doi.org/10.1016/j.jns.2014.03.049 0022-510X/© 2014 Elsevier B.V. All rights reserved.
ICH [7,8]. However, there is uncertainty over the effects of MMP9 genetic variations on ICH occurrence due to limitations of previous studies such as small sample size, limited variations of MMP9 gene investigated, mixed population, and non-Chinese population [9,10]. We aimed to explore the association of MMP9 genetic variation (alleles, genotypes and haplotype) with ICH susceptibility through a case–control study in Chinese Han population. 2. Materials and methods 2.1. Subjects Our case group consisted of consecutive Chinese Han patients (n = 181) with spontaneous ICH and hypertension who were admitted to West China Hospital and Nanjing First Hospital between Sep 2009 and Oct 2010. Clinical diagnosis of ICH was made according to the WHO criteria and was confirmed by brain CT or MRI scans [11]. Other hemorrhagic stroke such as subarachnoid hemorrhage (SAH), primary intraventricular hemorrhage, and secondary ICH due to traumatic, vascular structural abnormalities, antithrombotic therapy, or other known
56
J. Yang et al. / Journal of the Neurological Sciences 341 (2014) 55–57
causes were excluded. Deep location of ICH was defined as periventricular white matter, caudate, globus pallidus, putamen, internal capsule, and thalamus [12]. The control group consisted of 197 subjects with hypertension from the Health Examination Center of each participating hospital who were neurologically normal, and didn't have cerebrovascular and cardiovascular diseases as confirmed by medical history, physical examination, and laboratory examinations. Conventional risk factors included status of smoking and alcohol intake, presence of hypertension, diabetes and hypercholesterolemia. Participants who had smoked N 1 cigarette per day for more than one year were defined as current smokers. Patients who had consumed alcohol N50 ml per day for at least one year were classified as current drinkers. Hypertension was defined as systolic blood pressure (SBP) N 140 mmHg, diastolic blood pressure (DBP) N 90 mmHg or current use of antihypertensive agents. Diabetes was defined as fasting serum glucose level N 7.0 mmol/L or current use of antidiabetic drugs. Hypercholesterolemia was defined as fasting serum cholesterol level N 5.72 mmol/L or current use of lipid-lowering medicines. All participants involved in this study were unrelated Chinese Han and originated from a homogeneous population whose families must have resided for at least three generations in the same area of China. Peripheral blood leukocytes of patients and healthy controls were collected for genotype analysis. The study was approved by the Ethics Committees of each hospital. Participants were prospectively registered after gaining written informed consent from each subject or legal surrogate. 2.2. Genetic analysis MMP9 gene is 11,224 bp in length with 13 exons. Genomic regions containing 5 kb upstream and 5 kb downstream of MMP9 gene were screened for tagger single nucleotide polymorphisms (SNP) using the University of California Santa Cruz (UCSC) Genome Bioinformatics database (http://genome.ucsc.edu) and using data from the Hapmap Project (http://hapmap.ncbi.nlm.nih.gov), and with the settings of tagger pairwise, Minor Allele Frequency (MAF) N 0.1, r2 N 0.8 and Chinese Han Beijing population [13]. Genotyping was carried out at the Shanghai Benegene Biotechnology Co., LTD (Shanghai, China), employing MassARRAY technology platform (Sequenom, San Diego, USA) [14]. Haplotype analyses were performed using the softwares of Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview) and Unphased 3.1.7 (sites.google.com/site/fdudbridge/software) [13,15]. 2.3. Statistical analysis The Hardy–Weinberg equilibrium was assessed using a χ2 test with 1 degree of freedom. Statistical significance for allele, genotype and differences of haplotype frequencies between ICH patients and controls was assessed by the χ2 or Fisher's exact test for categorical variables, and T-Test or Mann–Whitney U tests were used for continuous variables. All the strength of association between the genetic variations of MMP9 and ICH was estimated with the odds ratio (OR) with 95% confidence interval (CI) by logistic regression model with Unphased 3.1.7 [15]. Bonferroni's test was used to correct for multiple testing. A p-value b 0.05 was considered statistically significant.
Table 2 Association between alleles of MMP9 and ICH susceptibility. SNP
Allele
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
rs3918241
A T C T A G C T A G A G
50/15% 292/85% 358/99% 4/1% 91/25% 271/75% 289/80% 73/20% 136/39% 212/61% 53/15% 309/85%
45/11% 349/89% 380/96% 14/4% 113/29% 279/71% 309/79% 81/21% 154/40% 234/60% 43/11% 351/89%
1.00 (reference) 0.75 (0.49–1.16) 1.00 (reference) & 1.00 (reference) 1.21 (0.87–1.67) 1.00 (reference) 0.96 (0.68–1.37) 1.00 (reference) 1.03 (0.76–1.38) 1.00 (reference) 0.71 (0.46–1.10)
rs1805088 rs17576 rs3918254 rs3787268 rs17577
Control (n = 197)
p
Age (year) Male Diabetes Hyperlipidemia Current drinker Current smoker
61.80 ± 13.27 136/75% 12/7% 5/3% 44/24% 47/26%
72.44 ± 12.71 109/55% 21/11% 14/7% 51/26% 53/27%
b0.01 0.54 0.20 0.06 0.81 0.91
ICH, intracerebral hemorrhage; Value are mean ± SD or n/%.
& 0.25 0.84 0.87 0.12
3. Results The demographic and clinical characteristics of the participants were summarized in Table 1. The 6 Tagger SNPs (rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577) within MMP9 gene were genotyped in 181 ICH patients and in 197 controls matched for hypertension. However, the ICH patients are younger than the controls (61.80 ± 13.27 vs. 72.44 ± 12.71 years, p b 0.05). More than 95.0% of the participants on case and control subjects were genotyped for the 6 Tagger SNPs, and 4 haplotype were successfully identified. All genotype distributions of MMP9 gene were in the Hardy–Weinberg equilibrium both in ICH and control groups (all p N 0.05). Table 2 showed the allele frequencies of ICH patients and controls. For the rs1805088, we could not perform statistical analysis due to the T allele's MAF b 0.05. For all the other alleles, there were no significant differences in frequencies between ICH patients and controls. The genotype frequencies of ICH patients and controls, and the association between the genotypes and ICH susceptibility in logistic regression analysis were illustrated in Table 3. There were no significant differences in frequencies between ICH patients and controls in all the genotypes except those with minor frequency b 0.05. Table 4 demonstrated the haplotypes frequencies of ICH patients and controls, and the association between the haplotypes and ICH susceptibility. With the 6 Tagger SNPs, four haplotypes were successfully genotyped on cases and controls. There were no significant differences in frequencies of the haplotypes between ICH patients and controls.
Table 3 Association between genotypes of MMP9 and ICH susceptibility. SNP
Genotypes
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
rs3918241
AA AT TT CC CT AA AG GG CC CT TT AA AG GG AA AG GG
4/2% 42/25% 125/73% 177/98% 4/2% 10/6% 71/39% 100/55% 113/62% 63/35% 5/3% 25/14% 86/49% 63/36% 5/3% 43/24% 133/37%
2/1% 41/21% 154/78% 183/93% 14/7% 1910/% 75/38% 102/52% 119/61% 71/36% 5/3% 30/15% 94/48% 70/36% 2/1% 39/20% 156/79%
1.00 (reference) & & 1.00 (reference) & 1.00 (reference) 1.80 (0.78–4.13) 1.86 (0.83–4.20) 1.00 (reference) 0.93 (0.61–1.43) & 1.00 (reference) 1.10 (0.60–2.01) 1.08 (0.57–2.03) 1.00 (reference) & &
rs1805088 rs17576
rs3918254
ICH (n = 181)
0.20
SNP, single nucleotide polymorphisms; ICH, intracerebral hemorrhage; reference, reference allele; 95% CI, 95% confidence interval; &, not suitable for statistical analysis due to minor allele frequency b.05.
Table 1 Demographic and clinical characteristics of the participants. Characteristics
p
rs3787268
rs17577
p & & & 0.85 0.53 0.75 & 0.85 0.98 & &
SNP, single nucleotide polymorphisms; ICH, intracerebral hemorrhage; reference, reference genotype; 95% CI, 95% confidence interval; &, not suitable for statistical analysis due to minor genotype frequency b0.05.
J. Yang et al. / Journal of the Neurological Sciences 341 (2014) 55–57 Table 4 Association between haplotypes of MMP9 and ICH susceptibility. SNPs
Haplotypes
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
p
123456 123456 123456 123456
ACGCGA TCACGG TCGCAG TCGTGG
45.99/14% 83.00/25% 127.00/38% 66.98/20%
43.00/11% 96.00/25% 152.00/78% 79.00/79%
1.00 (reference) 0.81 (0.49–1.35) 0.78 (0.48–1.26) 0.79 (0.47–1.34)
0.96 0.71 0.93
SNP, single nucleotide polymorphisms; 123456, rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577, respectively; ICH, intracerebral hemorrhage; reference, reference haplotype; 95% CI, 95% confidence interval.
4. Discussion We hypothesized that there would be differences in MMP9 genetic variations (allele, genotypes, and haplotypes) between ICH patients and control subjects. However, there were no significant association between MMP9 genetic variations and ICH susceptibility in the Chinese Han population. Firstly, our result suggested that the association between the alleles or genotypes of tagger SNPs within the MMP9 gene and ICH susceptibility is not significant, which is consistent with the results from previous studies on Caucasian population or mixed population [9,10]. However, the Caucasian study only involved 146 ICH patients and one SNP of MMP9 gene, and the mixed population study only involved 66 ICH patients without patients from the Chinese Han population, while our study included 181 ICH patients from the Chinese Han population, and explored all the Tagger SNPs of MMP9 gene further. Moreover, after stronger haplotype analyses (a method of retaining most of the information in a gene and reducing genotyping requirements) were performed, the association between the haplotypes of Tagger SNPs within the MMP9 gene and ICH susceptibility was still not significant, which is consistent with a previous study involving a small mixed population without Chinese Han patients [9]. Although previous studies showed that MMP9 could damage small vessel walls through degrading the extracellular matrix and may cause ICH [5,6], and MMP9 protein expression was also found to be influenced by genetic variations of MMP9 [16]. However, our results and previous studies indicated that there was no significant association between genetic variations of MMP9 and ICH susceptibility. The current study has several strengths, including the somewhat larger sample size than previous studies, as well as a case–control design to match for hypertension which is one of the most important risk factors of ICH. Furthermore, only Chinese Han patients with deep location of ICH were included, as superficial location of ICH may often be caused by other diseases such as vascular malformation and cerebrovascular amyloid angiopathy. In addition, comprehensive analyses of MMP9 genetic variations including both the genotypes and haplotypes, which are more powerful in exploring the association between MMP9 genetic variations and ICH occurrence. However, some limitations should also be noted. Firstly, control subjects were included for being naive of cerebrovascular and cardiovascular diseases by medical history, neurologically normal, and laboratory examinations. Without brain image trial, some control subjects might have been affected by silent stroke which could reduce the statistical power. Secondly, ICH is one of the complex and polygenic diseases. Although we explored the MMP9 genetic variations systematically, we might have ignored the interactions between MMP9 gene and other candidate genes for ICH. Thirdly, the ICH cases were younger than
57
control subjects, which could have led to a reduction in statistical power in detecting MMP9 gene's impact on ICH susceptibility. In summary, the MMP9 genetic variations were not significantly associated with ICH susceptibility in the Chinese Han population. Conflict of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by grants (30900472, 81100859) from the National Natural Science Foundation of China, a grant (2012M511302) from China Postdoctoral Science Foundation, and a grant (201208008) from Nanjing Health Bureau. The sponsors of the grants had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. We thank the patients who participated in this trial and their relatives; the clinical and research teams of the neurology and neurosurgery departments; Miss Anne Jian from the University of Melbourne for her help with English writing; and Dr. Zhi Xu from Southeast University for her help with statistical analysis. References [1] Krishnamurthi Rita V, VLF, Forouzanfar Mohammad H, Mensah George A, Connor Myles, Bennett Derrick A. Global and regional burden of first-ever ischaemic and haemorrhagic stroke during 1990–2010: findings from the Global Burden of Disease Study 2010. Lancet Glob Health 2013;1:e259–81. [2] van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ. Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol 2010;9:167–76. [3] Morgenstern LB, Hemphill 3rd JC, Anderson C, Becker K, Broderick JP, Connoly Jr ES. Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/ American Stroke Association. Stroke 2010;41:2108–29. [4] Qureshi AI, Mendelow AD, Hanley DF. Intracerebral haemorrhage. Lancet 2009;373:1632–44. [5] Lo EH. A new penumbra: transitioning from injury into repair after stroke. Nat Med 2008;14:497–500. [6] Dumont O, Loufrani L, Henrion D. Key role of the NO-pathway and matrix metalloprotease-9 in high blood flow-induced remodeling of rat resistance arteries. Arterioscler Thromb Vasc Biol 2007;27:317–24. [7] Inzitari D, Giusti B, Nencini P, Gori AM, Nesi M, Palumbo V, et al. MMP9 variation after thrombolysis is associated with hemorrhagic transformation of lesion and death. Stroke 2013;44:2901–3. [8] Brunswick AS, Hwang BY, Appelboom G, Hwang RY, Piazza MA, Connolly Jr ES. Serum biomarkers of spontaneous intracerebral hemorrhage induced secondary brain injury. J Neurol Sci 2012;321:1–10. [9] Kaplan RC, Smith NL, Zucker S, Heckbert SR, Rice K, Psaty BM. Matrix metalloproteinase-3 (MMP3) and MMP9 genes and risk of myocardial infarction, ischemic stroke, and hemorrhagic stroke. Atherosclerosis 2008;201:130–7. [10] Szczudlik P, Borratynska A. Association between the −1562 C/T MMP-9 polymorphism and cerebrovascular disease in a Polish population. Neurol Neurochir Pol 2010;44:350–7. [11] Special report from the World-Health-Organization — stroke — 1989 — recommendations on stroke prevention, diagnosis, and therapy. Stroke 1989;20:1407–31. [12] Flaherty ML, Woo D, Haverbusch M, Sekar P, Khoury L, Sauerbeck L, et al. Racial variations in location and risk of intracerebral hemorrhage. Stroke 2005;36:934–7. [13] Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics (Oxford, England) 2005;21:263–5. [14] Cunninghame Graham DS, Graham RR, Manku H, Wong AK, Whittaker JC, Gaffney PM, et al. Polymorphism at the TNF superfamily gene TNFSF4 confers susceptibility to systemic lupus erythematosus. Nat Genet 2008;40:83–9. [15] Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Hum Hered 2008;66:87–98. [16] Medley TL, Cole TJ, Dart AM, Gatzka CD, Kingwell BA. Matrix metalloproteinase-9 genotype influences large artery stiffness through effects on aortic gene and protein expression. Arterioscler Thromb Vasc Biol 2004;24:1479–84.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Genetic variations of MMP9 gene and intracerebral hemorrhage susceptibility: A case–control study in Chinese Han population Jie Yang a,b,1, Bo Wu a,1, Sen Lin a, Junshan Zhou b, Yingbin Li c, Wei Dong a, Hisatomi Arima d, Chanfei Zhang a, Yukai Liu b, Ming Liu a,⁎, for the Chengdu stroke registry and Nanjing First Hospital stroke registry investigators a
Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, China Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Neurosurgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China d The George Institute for Global Health, Royal Prince Alfred Hospital and University of Sydney, Sydney, NSW 2050, Australia b c
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 23 March 2014 Accepted 25 March 2014 Available online 2 April 2014 Keywords: Matrix metalloproteinase 9 Single nucleotide polymorphism Genotype Haplotype Intracerebral hemorrhage Etiology
a b s t r a c t Objective: To investigate the association between genetic variations of matrix metalloproteinase 9 (MMP9) gene and intracerebral hemorrhage (ICH) susceptibility in Chinese Han population. Methods: The clinical data and peripheral blood samples from the patients with ICH and hypertension, and controlled subjects with hypertension only, were collected. MassARRAY Analyzer was used to genotype the tagger single nucleotide polymorphism (SNP) of MMP9 gene. Haploview4.2 and Unphased3.1.7 were employed to construct haplotypes and to analyze the association between genetic variations (alleles, genotypes and haplotypes) of MMP9 gene and ICH susceptibility. Results: 181 patients with ICH and hypertension, and 197 patients with hypertension only, were recruited between Sep 2009 and Oct 2010. Patients in the ICH group were younger (61.80 ± 13.27 vs. 72.44 ± 12.71 years, p b 0.05). Other conventional risk factors between the ICH and control groups were similar. There were 6 Tagger SNPs and 4 haplotypes of MMP9 gene in our sample population. Our logistical regression analysis showed that there were no significant associations between genetic variations of the MPP9 gene and ICH susceptibility (all p N 0.05). Conclusions: The genetic variations of MMP9 gene were not significantly associated with ICH susceptibility in the Chinese Han population. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute spontaneous intracerebral hemorrhage (ICH) is estimated to occur in ≈5 million people worldwide each year, and most of whom either die or are left seriously disabled because of limited effective treatment strategies available [1–3]. Prevention of ICH requires better knowledge on genetic susceptibility as well as on conventional risk factors. Most primary ICH originates from bleeding at small arteries, which are characterized by breakage of extracellular matrix in arterial walls [4]. Matrix metalloproteinase 9 (MMP9) can damage small vessels through degrading the extracellular matrix and may cause ICH [5,6]. Increased plasma MMP9 levels were also observed in patients with
⁎ Corresponding author at: Stroke Clinical Research Unit, Department of Neurology, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Chengdu 610041, China. Tel.: +86 28 8181 2671; fax: +86 28 8542 3551. E-mail address: [email protected] (M. Liu). 1 The first two authors should be regarded as co-first authors.
http://dx.doi.org/10.1016/j.jns.2014.03.049 0022-510X/© 2014 Elsevier B.V. All rights reserved.
ICH [7,8]. However, there is uncertainty over the effects of MMP9 genetic variations on ICH occurrence due to limitations of previous studies such as small sample size, limited variations of MMP9 gene investigated, mixed population, and non-Chinese population [9,10]. We aimed to explore the association of MMP9 genetic variation (alleles, genotypes and haplotype) with ICH susceptibility through a case–control study in Chinese Han population. 2. Materials and methods 2.1. Subjects Our case group consisted of consecutive Chinese Han patients (n = 181) with spontaneous ICH and hypertension who were admitted to West China Hospital and Nanjing First Hospital between Sep 2009 and Oct 2010. Clinical diagnosis of ICH was made according to the WHO criteria and was confirmed by brain CT or MRI scans [11]. Other hemorrhagic stroke such as subarachnoid hemorrhage (SAH), primary intraventricular hemorrhage, and secondary ICH due to traumatic, vascular structural abnormalities, antithrombotic therapy, or other known
56
J. Yang et al. / Journal of the Neurological Sciences 341 (2014) 55–57
causes were excluded. Deep location of ICH was defined as periventricular white matter, caudate, globus pallidus, putamen, internal capsule, and thalamus [12]. The control group consisted of 197 subjects with hypertension from the Health Examination Center of each participating hospital who were neurologically normal, and didn't have cerebrovascular and cardiovascular diseases as confirmed by medical history, physical examination, and laboratory examinations. Conventional risk factors included status of smoking and alcohol intake, presence of hypertension, diabetes and hypercholesterolemia. Participants who had smoked N 1 cigarette per day for more than one year were defined as current smokers. Patients who had consumed alcohol N50 ml per day for at least one year were classified as current drinkers. Hypertension was defined as systolic blood pressure (SBP) N 140 mmHg, diastolic blood pressure (DBP) N 90 mmHg or current use of antihypertensive agents. Diabetes was defined as fasting serum glucose level N 7.0 mmol/L or current use of antidiabetic drugs. Hypercholesterolemia was defined as fasting serum cholesterol level N 5.72 mmol/L or current use of lipid-lowering medicines. All participants involved in this study were unrelated Chinese Han and originated from a homogeneous population whose families must have resided for at least three generations in the same area of China. Peripheral blood leukocytes of patients and healthy controls were collected for genotype analysis. The study was approved by the Ethics Committees of each hospital. Participants were prospectively registered after gaining written informed consent from each subject or legal surrogate. 2.2. Genetic analysis MMP9 gene is 11,224 bp in length with 13 exons. Genomic regions containing 5 kb upstream and 5 kb downstream of MMP9 gene were screened for tagger single nucleotide polymorphisms (SNP) using the University of California Santa Cruz (UCSC) Genome Bioinformatics database (http://genome.ucsc.edu) and using data from the Hapmap Project (http://hapmap.ncbi.nlm.nih.gov), and with the settings of tagger pairwise, Minor Allele Frequency (MAF) N 0.1, r2 N 0.8 and Chinese Han Beijing population [13]. Genotyping was carried out at the Shanghai Benegene Biotechnology Co., LTD (Shanghai, China), employing MassARRAY technology platform (Sequenom, San Diego, USA) [14]. Haplotype analyses were performed using the softwares of Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview) and Unphased 3.1.7 (sites.google.com/site/fdudbridge/software) [13,15]. 2.3. Statistical analysis The Hardy–Weinberg equilibrium was assessed using a χ2 test with 1 degree of freedom. Statistical significance for allele, genotype and differences of haplotype frequencies between ICH patients and controls was assessed by the χ2 or Fisher's exact test for categorical variables, and T-Test or Mann–Whitney U tests were used for continuous variables. All the strength of association between the genetic variations of MMP9 and ICH was estimated with the odds ratio (OR) with 95% confidence interval (CI) by logistic regression model with Unphased 3.1.7 [15]. Bonferroni's test was used to correct for multiple testing. A p-value b 0.05 was considered statistically significant.
Table 2 Association between alleles of MMP9 and ICH susceptibility. SNP
Allele
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
rs3918241
A T C T A G C T A G A G
50/15% 292/85% 358/99% 4/1% 91/25% 271/75% 289/80% 73/20% 136/39% 212/61% 53/15% 309/85%
45/11% 349/89% 380/96% 14/4% 113/29% 279/71% 309/79% 81/21% 154/40% 234/60% 43/11% 351/89%
1.00 (reference) 0.75 (0.49–1.16) 1.00 (reference) & 1.00 (reference) 1.21 (0.87–1.67) 1.00 (reference) 0.96 (0.68–1.37) 1.00 (reference) 1.03 (0.76–1.38) 1.00 (reference) 0.71 (0.46–1.10)
rs1805088 rs17576 rs3918254 rs3787268 rs17577
Control (n = 197)
p
Age (year) Male Diabetes Hyperlipidemia Current drinker Current smoker
61.80 ± 13.27 136/75% 12/7% 5/3% 44/24% 47/26%
72.44 ± 12.71 109/55% 21/11% 14/7% 51/26% 53/27%
b0.01 0.54 0.20 0.06 0.81 0.91
ICH, intracerebral hemorrhage; Value are mean ± SD or n/%.
& 0.25 0.84 0.87 0.12
3. Results The demographic and clinical characteristics of the participants were summarized in Table 1. The 6 Tagger SNPs (rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577) within MMP9 gene were genotyped in 181 ICH patients and in 197 controls matched for hypertension. However, the ICH patients are younger than the controls (61.80 ± 13.27 vs. 72.44 ± 12.71 years, p b 0.05). More than 95.0% of the participants on case and control subjects were genotyped for the 6 Tagger SNPs, and 4 haplotype were successfully identified. All genotype distributions of MMP9 gene were in the Hardy–Weinberg equilibrium both in ICH and control groups (all p N 0.05). Table 2 showed the allele frequencies of ICH patients and controls. For the rs1805088, we could not perform statistical analysis due to the T allele's MAF b 0.05. For all the other alleles, there were no significant differences in frequencies between ICH patients and controls. The genotype frequencies of ICH patients and controls, and the association between the genotypes and ICH susceptibility in logistic regression analysis were illustrated in Table 3. There were no significant differences in frequencies between ICH patients and controls in all the genotypes except those with minor frequency b 0.05. Table 4 demonstrated the haplotypes frequencies of ICH patients and controls, and the association between the haplotypes and ICH susceptibility. With the 6 Tagger SNPs, four haplotypes were successfully genotyped on cases and controls. There were no significant differences in frequencies of the haplotypes between ICH patients and controls.
Table 3 Association between genotypes of MMP9 and ICH susceptibility. SNP
Genotypes
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
rs3918241
AA AT TT CC CT AA AG GG CC CT TT AA AG GG AA AG GG
4/2% 42/25% 125/73% 177/98% 4/2% 10/6% 71/39% 100/55% 113/62% 63/35% 5/3% 25/14% 86/49% 63/36% 5/3% 43/24% 133/37%
2/1% 41/21% 154/78% 183/93% 14/7% 1910/% 75/38% 102/52% 119/61% 71/36% 5/3% 30/15% 94/48% 70/36% 2/1% 39/20% 156/79%
1.00 (reference) & & 1.00 (reference) & 1.00 (reference) 1.80 (0.78–4.13) 1.86 (0.83–4.20) 1.00 (reference) 0.93 (0.61–1.43) & 1.00 (reference) 1.10 (0.60–2.01) 1.08 (0.57–2.03) 1.00 (reference) & &
rs1805088 rs17576
rs3918254
ICH (n = 181)
0.20
SNP, single nucleotide polymorphisms; ICH, intracerebral hemorrhage; reference, reference allele; 95% CI, 95% confidence interval; &, not suitable for statistical analysis due to minor allele frequency b.05.
Table 1 Demographic and clinical characteristics of the participants. Characteristics
p
rs3787268
rs17577
p & & & 0.85 0.53 0.75 & 0.85 0.98 & &
SNP, single nucleotide polymorphisms; ICH, intracerebral hemorrhage; reference, reference genotype; 95% CI, 95% confidence interval; &, not suitable for statistical analysis due to minor genotype frequency b0.05.
J. Yang et al. / Journal of the Neurological Sciences 341 (2014) 55–57 Table 4 Association between haplotypes of MMP9 and ICH susceptibility. SNPs
Haplotypes
ICH (n/%)
Controls (n/%)
Odds ratio (95% CI)
p
123456 123456 123456 123456
ACGCGA TCACGG TCGCAG TCGTGG
45.99/14% 83.00/25% 127.00/38% 66.98/20%
43.00/11% 96.00/25% 152.00/78% 79.00/79%
1.00 (reference) 0.81 (0.49–1.35) 0.78 (0.48–1.26) 0.79 (0.47–1.34)
0.96 0.71 0.93
SNP, single nucleotide polymorphisms; 123456, rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577, respectively; ICH, intracerebral hemorrhage; reference, reference haplotype; 95% CI, 95% confidence interval.
4. Discussion We hypothesized that there would be differences in MMP9 genetic variations (allele, genotypes, and haplotypes) between ICH patients and control subjects. However, there were no significant association between MMP9 genetic variations and ICH susceptibility in the Chinese Han population. Firstly, our result suggested that the association between the alleles or genotypes of tagger SNPs within the MMP9 gene and ICH susceptibility is not significant, which is consistent with the results from previous studies on Caucasian population or mixed population [9,10]. However, the Caucasian study only involved 146 ICH patients and one SNP of MMP9 gene, and the mixed population study only involved 66 ICH patients without patients from the Chinese Han population, while our study included 181 ICH patients from the Chinese Han population, and explored all the Tagger SNPs of MMP9 gene further. Moreover, after stronger haplotype analyses (a method of retaining most of the information in a gene and reducing genotyping requirements) were performed, the association between the haplotypes of Tagger SNPs within the MMP9 gene and ICH susceptibility was still not significant, which is consistent with a previous study involving a small mixed population without Chinese Han patients [9]. Although previous studies showed that MMP9 could damage small vessel walls through degrading the extracellular matrix and may cause ICH [5,6], and MMP9 protein expression was also found to be influenced by genetic variations of MMP9 [16]. However, our results and previous studies indicated that there was no significant association between genetic variations of MMP9 and ICH susceptibility. The current study has several strengths, including the somewhat larger sample size than previous studies, as well as a case–control design to match for hypertension which is one of the most important risk factors of ICH. Furthermore, only Chinese Han patients with deep location of ICH were included, as superficial location of ICH may often be caused by other diseases such as vascular malformation and cerebrovascular amyloid angiopathy. In addition, comprehensive analyses of MMP9 genetic variations including both the genotypes and haplotypes, which are more powerful in exploring the association between MMP9 genetic variations and ICH occurrence. However, some limitations should also be noted. Firstly, control subjects were included for being naive of cerebrovascular and cardiovascular diseases by medical history, neurologically normal, and laboratory examinations. Without brain image trial, some control subjects might have been affected by silent stroke which could reduce the statistical power. Secondly, ICH is one of the complex and polygenic diseases. Although we explored the MMP9 genetic variations systematically, we might have ignored the interactions between MMP9 gene and other candidate genes for ICH. Thirdly, the ICH cases were younger than
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control subjects, which could have led to a reduction in statistical power in detecting MMP9 gene's impact on ICH susceptibility. In summary, the MMP9 genetic variations were not significantly associated with ICH susceptibility in the Chinese Han population. Conflict of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by grants (30900472, 81100859) from the National Natural Science Foundation of China, a grant (2012M511302) from China Postdoctoral Science Foundation, and a grant (201208008) from Nanjing Health Bureau. The sponsors of the grants had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. We thank the patients who participated in this trial and their relatives; the clinical and research teams of the neurology and neurosurgery departments; Miss Anne Jian from the University of Melbourne for her help with English writing; and Dr. Zhi Xu from Southeast University for her help with statistical analysis. References [1] Krishnamurthi Rita V, VLF, Forouzanfar Mohammad H, Mensah George A, Connor Myles, Bennett Derrick A. Global and regional burden of first-ever ischaemic and haemorrhagic stroke during 1990–2010: findings from the Global Burden of Disease Study 2010. Lancet Glob Health 2013;1:e259–81. [2] van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ. Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol 2010;9:167–76. [3] Morgenstern LB, Hemphill 3rd JC, Anderson C, Becker K, Broderick JP, Connoly Jr ES. Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/ American Stroke Association. Stroke 2010;41:2108–29. [4] Qureshi AI, Mendelow AD, Hanley DF. Intracerebral haemorrhage. Lancet 2009;373:1632–44. [5] Lo EH. A new penumbra: transitioning from injury into repair after stroke. Nat Med 2008;14:497–500. [6] Dumont O, Loufrani L, Henrion D. Key role of the NO-pathway and matrix metalloprotease-9 in high blood flow-induced remodeling of rat resistance arteries. Arterioscler Thromb Vasc Biol 2007;27:317–24. [7] Inzitari D, Giusti B, Nencini P, Gori AM, Nesi M, Palumbo V, et al. MMP9 variation after thrombolysis is associated with hemorrhagic transformation of lesion and death. Stroke 2013;44:2901–3. [8] Brunswick AS, Hwang BY, Appelboom G, Hwang RY, Piazza MA, Connolly Jr ES. Serum biomarkers of spontaneous intracerebral hemorrhage induced secondary brain injury. J Neurol Sci 2012;321:1–10. [9] Kaplan RC, Smith NL, Zucker S, Heckbert SR, Rice K, Psaty BM. Matrix metalloproteinase-3 (MMP3) and MMP9 genes and risk of myocardial infarction, ischemic stroke, and hemorrhagic stroke. Atherosclerosis 2008;201:130–7. [10] Szczudlik P, Borratynska A. Association between the −1562 C/T MMP-9 polymorphism and cerebrovascular disease in a Polish population. Neurol Neurochir Pol 2010;44:350–7. [11] Special report from the World-Health-Organization — stroke — 1989 — recommendations on stroke prevention, diagnosis, and therapy. Stroke 1989;20:1407–31. [12] Flaherty ML, Woo D, Haverbusch M, Sekar P, Khoury L, Sauerbeck L, et al. Racial variations in location and risk of intracerebral hemorrhage. Stroke 2005;36:934–7. [13] Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics (Oxford, England) 2005;21:263–5. [14] Cunninghame Graham DS, Graham RR, Manku H, Wong AK, Whittaker JC, Gaffney PM, et al. Polymorphism at the TNF superfamily gene TNFSF4 confers susceptibility to systemic lupus erythematosus. Nat Genet 2008;40:83–9. [15] Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Hum Hered 2008;66:87–98. [16] Medley TL, Cole TJ, Dart AM, Gatzka CD, Kingwell BA. Matrix metalloproteinase-9 genotype influences large artery stiffness through effects on aortic gene and protein expression. Arterioscler Thromb Vasc Biol 2004;24:1479–84.
