JNS-13198; No of Pages 4 Journal of the Neurological Sciences xxx (2014) xxx–xxx
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Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population Jie Yang a,b,1, Sen Lin a,1, Junshan Zhou b, Bo Wu a, Wei Dong a, Hisatomi Arima c, Hua Liu d, Jing Zhang a, Jie Li a, 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 The George Institute for Global Health, Royal Prince Alfred Hospital, University of Sydney, Sydney, NSW 2050, Australia d Department of Neurology, The Third People's Hospital of Mianyang, Luzhou Medical College, Mianyang 621000, China b c
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 20 April 2014 Accepted 12 May 2014 Available online xxxx Keywords: Matrix metalloproteinase 9 Genetic variation Single nucleotide polymorphism Haplotype Intracerebral hemorrhage Outcome
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) outcome in Chinese Han population. Methods: The clinical data and peripheral blood samples from the patients with ICH were collected. The patients were followed up for 3 months, and poor outcome was defined as death or dependency (modified Rankin scale score of 3–6). MassARRAY Analyser was used to genotype the tagger single nucleotide polymorphisms (SNPs) of MMP9 gene. Construction of haplotypes and genetic comparisons were performed by employing PLINK 1.0.7 software. Results: 181 patients with ICH were recruited between September 2009 and October 2010. Information on genetic variations and follow-up assessments were available for 169 (93.4%) patients. Independent patients at 90 days were younger than those who died or dependent (57.82 ± 13.47 vs. 66.99 ± 11.49 years, p b 0.01). In addition, independent patients had lower National Institutes of Health Stroke Scale (NIHSS) score (4[1–6] vs. 13[9–19], p b 0.01). There were no significant associations (all p N 0.05) between MMP9 genetic variations (alleles, genotypes and haplotypes) and ICH outcome after adjustment for conventional risk factors. Conclusions: The genetic variations of MMP9 gene were not significantly associated with ICH outcome at 90 days in 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]. Better management of ICH requires better knowledge on genetic as well as on conventional risk factors of poor prognosis. Brain injury after ICH involves different mechanisms such as physical disruption and mass effect due to hematoma and cerebral edema, and secondary adverse effects of coagulation cascade, hemoglobin breakdown products, and inflammation [4]. Among these, hematoma volume is one of the most important determinants of poor outcome in ICH [5]. Development of perihematomal cerebral edema also leads to subsequent poor clinical outcomes [6,7]. Studies indicated that matrix
⁎ 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 These authors contributed equally to this work.
metalloproteinase 9 (MMP9) levels increased in plasma samples of patients with ICH [8,9]. Increased plasma MMP9 levels were also found to be associated with hematoma growth and perihematomal cerebral edema both in ICH animal [10,11] and in ICH patients [12,13]. Moreover, MMP9 level was predicted by its genetic variations [14]. However, there is no previous report on whether MMP9 genetic variations predict poor prognosis after ICH. We aimed to systematically explore the association between MMP9 genetic variations (alleles, genotypes and haplotypes) and ICH outcome through a larger sample cohort study in Chinese Han population. 2. Materials and methods 2.1. Subjects Our cohort consisted of consecutive Chinese Han patients (n = 181) experiencing a deep location of ICH with hypertension who admitted to West China Hospital or Nanjing First Hospital between September 2009 and October 2010. Clinical diagnosis of ICH was made according to the WHO criteria and was confirmed by brain CT or MRI scans [15]. Other hemorrhagic strokes such as subarachnoid hemorrhage, primary
http://dx.doi.org/10.1016/j.jns.2014.05.021 0022-510X/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
2
J. Yang et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
2.3. Follow-up and outcome
Table 1 Demographic and clinical characteristics of the participants. Characteristics
Death or dependency (n = 74)
Independency (n = 95)
p
Age (year) Male SBP (mmHg) DBP (mmHg) GCS score, median (IQR) NIHSS score, median (IQR) Hypertension Diabetes Hyperlipidemia Stroke Current drinker Current smoker
66.99 ± 11.49 57/77.0% 166.92 ± 25.83 95.16 ± 16.02 13 (8–15) 13 (9–19) 65/87.8% 6/8.1% 2/2.7% 7/9.5% 19/25.7% 18/24.3%
57.82 ± 13.47 68/71.6% 166.99 ± 28.76 100.07 ± 19.26 15 (14–15) 4 (1–6) 80/84.2% 6/6.3% 3/3.2% 6/6.3% 25/26.3% 29/30.5%
b0.01 0.48 0.99 0.08 b0.01 b0.01 0.66 0.77 1.00 0.56 1.00 0.39
SBP, systolic blood pressure; DBP, diastolic blood pressure; GCS, Glasgow Coma Scale; NIHSS, National Institutes of Health Stroke Scale; values are mean ± SD, median (IQR) or n/%.
intraventricular hemorrhage, and ICH due to traumatic, vascular structural abnormalities, antithrombotic therapy, or other known secondary causes were excluded. Deep location of ICH was defined as periventricular white matter, caudate, globus pallidus, putamen, internal capsule, and thalamus [16]. 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 were collected. Detailed information about patient demography (age and gender), conventional risk factors, and stroke severity were collected at baseline. Severity of stroke was evaluated with the Glasgow Coma Scale (GCS) and the National Institutes of Health Stroke Scale (NIHSS) [17,18]. This study was approved by the Ethics Committees of each hospital. Participants were prospectively registered and followed up 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 (SNPs) 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 00.1, r2 N 0.8 and Chinese Han Beijing population [19]. Genotyping was carried out at the Shanghai Benegene Biotechnology Co., LTD (Shanghai, China. http://www. benegene.com.cn/snp/english_site/index.html), using MassARRAY technology platform (Sequenom, San Diego, USA). Haplotypes were identified using PLINK 1.07 (http://pngu.mgh.harvard.edu/purcell/ plink).
The participants were prospectively followed up at 3 months after ICH. The primary outcome was death or dependency at 3 months after ICH. Death was defined as the cumulative all-cause death. Dependency was defined as modified Rankin scale (mRS) score of 3–5 [20].
2.4. Statistical analysis Binary data were described as percentages while continuous data were expressed as the mean ± standard deviation (SD). The Hardy– Weinberg equilibrium was assessed using a χ2 test with 1 degree of freedom. Three genetic models (allelic comparison, dominant and recessive model) were assumed while performing analyses of the distribution of alleles and genotypes between groups. After adjustment for gender, age, and baseline NIHSS score, and controlling for multiple tests with the number of SNPs genotyped, statistical significance of allele, genotypes and haplotypes on ICH outcome was assessed by logistic regression with Plink 1.0.7. A p-value b 0.05 (two-sided) was considered statistically significant.
3. Results The demographic and clinical characteristics of the participants were summarized in Table 1. At the end of 3 months, DNA data and follow-up data were available for 169 (93.4%) patients. Independent patients at 90 days were younger than those dead or dependent (57.82 ± 13.47 vs. 66.99 ± 11.49 years, p b 0.01). In addition, independent patients had higher GCS (15[14, 15] vs. 13[8–15], p b 0.01) and lower NIHSS scores (4[1–6] vs. 13[9–19], p b 0.01). The 6 tagger SNPs (rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577) within MMP9 gene were genotyped in those ICH patients. More than 95.0% participants were genotyped for the 6 tagger SNPs, and 4 haplotypes were successfully identified. Distribution of all genotypes was in the Hardy–Weinberg equilibrium in this cohort (p N 0.05). Table 2 showed the allele frequencies in ICH patients. After adjustment for conventional risk factors (gender, age, and baseline NIHSS score) and multiple tests, there were no significant differences in frequencies between dead or dependent patients and independent ones (all p N 0.05). Table 3 illustrated the genotype frequencies in ICH patients. After adjustment for the same conventional risk factors and multiple tests, there was no significant association between genotypes of MMP9 gene and death or dependency in dominant or recessive models (all p N 0.05). Table 4 demonstrated the haplotype frequencies in ICH patients. Four haplotypes were constructed with the 6 tagger SNPs of MMP9 gene. There was no significant association between the haplotypes and death or dependency after adjustment for the same conventional risk factors and multiple tests (all p N 0.05).
Table 2 Association between alleles of MMP9 and ICH independency at 90 days. SNPs
A1/A2
Death or dependency (n)
Independency (n)
Odds ratio
pa
pb
rs3918241 rs1805088 rs17576 rs3918254 rs3787268 rs17577
A/T T/C A/G T/C A/G A/G
18/124 2/146 41/107 23/125 62/82 19/129
27/149 2/188 47/143 44/146 64/116 29/161
1.77 0.38 1.20 0.53 1.05 1.64
0.30 0.54 0.63 0.18 0.91 0.32
0.99 0.99 0.99 0.99 0.99 0.99
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; A1, allele 1; A2, allele 2. a Adjusted by gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
J. Yang et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
3
Table 3 Association between genotypes of MMP9 and ICH independency at 90 days. SNPs
Genotypes
Death or dependency (n)
Independency (n)
Comparison models
Odds ratio
pa
pb
rs3918241
AA AT TT CC CT TT AA AG GG CC CT TT AA AG GG AA AG GG
2 14 55 72 2 0 2 37 35 51 23 0 11 40 21 2 15 57
2 23 63 93 2 0 8 31 56 55 36 4 12 40 38 3 23 69
Recessive
AA/(AT + TT)
Dominant Dominant
TT/(AT + AA) CC/(CT + TT)
Recessive Recessive
TT/(CT + CC) AA/(AG + GG)
Dominant Dominant
GG/(AG + AA) CC/(CT + TT)
Recessive Recessive
TT/(CT + CC) AA/(AG + GG)
Dominant Recessive
GG/(AG + AA) AA/(AG + GG)
Dominant
GG/(AG + AA)
18.65 – 1.38 0.38 – & 0.10 – 2.236 0.53 – b0.01 0.62 – 1.40 7.37 – 1.42
0.04 – 0.59 0.54 – & 0.04 – 0.09 0.19 – 1.00 0.50 – 0.52 0.12 – 0.54
0.24 – 0.99 N1 – & 0.24 – 0.99 0.99 – 0.99 0.99 – 0.99 0.72 – 0.99
rs1805088
rs17576
rs3918254
rs3787268
rs17577
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; &, not suitable for statistical analysis due to minor genotype frequency b0.05. a Adjusted for gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
Table 4 Association between haplotypes of MMP9 and ICH independency at 90 days. SNPs
Haplotypes
Death or dependency (%)
Independency (%)
Odds ratio
pa
pb
123456 123456 123456 123456
ACGCGA TCACGG TCGCAG TCGTGG
0.127 0.275 0.444 0.155
0.1534 0.250 0.368 0.229
1.72 1.14 1.04 0.64
0.29 0.73 0.90 0.35
0.99 0.99 0.99 0.99
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; 123456, indicate rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577, respectively. a Adjusted by gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
4. Discussion We hypothesized that there would be roles of MMP9 genetic variations (alleles, genotypes and haplotypes) on ICH outcome. However, there was no significant association between MMP9 genetic variations and death or dependency at 90 days after ICH onset in the present Chinese Han population. The human MMP9 gene was mapped to chromosome 20q11.2-13.1. Its product is a gelatinase B that fulfills the role of degrading the extracellular matrix both in the vessel and in the brain, and which exacerbate the hematoma growth and cerebral edema after ICH [10–13,21]. The MMP9 gene variations had been proposed to be implicated in poor outcome after ischemic stroke in Caucasian population [8]. However, recovery processes after ischemic stroke and hemorrhagic stroke may be different which led us to explore whether MMP9 gene variations are associated with ICH outcome [22]. To the best of our knowledge, we first systematically explored the association between MMP9 genetic variations and ICH outcomes in a larger (n = 169) sample of Chinese Han population. However, our results indicated that there was no significant association between genetic variations of MMP9 and ICH outcome even after more powerful haplotype analysis (a method of retaining most of the information in a gene and with reducing genotyping requirements) [23]. The current study has several strengths, including the homogeneity sample in the same Chinese Han population. In addition, only patients with deep location of ICH were included because superficial location of ICH maybe often caused by other reasons such as vascular malformation and cerebrovascular amyloid angiopathy. Furthermore, comprehensive analyses of MMP9 genetic variations include the alleles, genotypes and haplotypes, which are more powerful to explore the association between MMP9 genetic variations and ICH outcome [23]. Some limitations should also be noted. First, ICH is one of the complex and polygenic diseases. Although we explored the MMP9 genetic
variations systematically, we might ignore the interaction between MMP9 gene and other candidate genes for ICH outcome. Gene–gene interactions should be considered in future studies. Second, some frequencies of the genetic variants were under 5%, which may lead to insufficient statistical power of our analyses. Furthermore, there was no information on ICH hematoma volume in this registry. So we did not know if the hematoma volume had influenced our results. However, our multivariable analysis included admission NIHSS score as a covariate, and we believe that lack of information on hematoma volume does not completely invalidate the findings of the present analysis. In summary, the MMP9 genetic variations were not significantly associated with ICH outcome in the present Chinese Han population. Future studies should be performed with larger samples and among other ethnic populations, with gene–gene interactions being considered. Conflict of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by grants 30900472 and 81100859 from the National Natural Science Foundation of China, grant 2012M511302 from China Postdoctoral Science Foundation, and project 201208008 from Nanjing Health Bureau. We thank Dr. Xiang Liu from West China School of Public Health, Sichuan University for his statistical help. References [1] Krishnamurthi VLF Rita V, 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.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
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[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 JC, Anderson C, et al. 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] Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol 2012;11:720–31. [5] Broderick JP, Brott TG, Duldner JE, Tomsick T, Huster G. Volume of intracerebral hemorrhage. A powerful and easy-to-use predictor of 30-day mortality. Stroke 1993;24:987–93. [6] Zazulia AR, Diringer MN, Derdeyn CP, Powers WJ. Progression of mass effect after intracerebral hemorrhage. Stroke 1999;30:1167–73. [7] Inaji M, Tomita H, Tone O, Tamaki M, Suzuki R, Ohno K. Chronological changes of perihematomal edema of human intracerebral hematoma. Acta Neurochir Suppl 2003;86:445–8. [8] Inzitari D, Giusti B, Nencini P, et al. MMP9 variation after thrombolysis is associated with hemorrhagic transformation of lesion and death. Stroke 2013;44:2901–3. [9] Brunswick AS, Hwang BY, Appelboom G, Hwang RY, Piazza MA, Connolly ES. Serum biomarkers of spontaneous intracerebral hemorrhage induced secondary brain injury. J Neurol Sci 2012;321:1–10. [10] Barr TL, Latour LL, Lee KY, et al. Blood–brain barrier disruption in humans is independently associated with increased matrix metalloproteinase-9. Stroke 2010;41: E123–8. [11] Silva Y, Leira R, Tejada J, et al. Molecular signatures of vascular injury are associated with early growth of intracerebral hemorrhage. Stroke 2005;36:86–91.
[12] Wang J, Tsirka SE. Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage. Brain 2005;128:1622–33. [13] Tejima E, Zhao BQ, Tsuji K, et al. Astrocytic induction of matrix metalloproteinase-9 and edema in brain hemorrhage. J Cereb Blood Flow Metab 2007;27:460–8. [14] Yuan M, Zhan Q, Duan X, et al. A functional polymorphism at miR-491-5p binding site in the 3′-UTR of MMP-9 gene confers increased risk for atherosclerotic cerebral infarction in a Chinese population. Atherosclerosis 2013;226:447–52. [15] Special Report from the World-Health-Organization — stroke — 1989 — recommendations on stroke prevention, diagnosis, and therapy. Stroke 1989;20:1407–31. [16] Flaherty ML, Woo D, Haverbusch M, et al. Racial variations in location and risk of intracerebral hemorrhage. Stroke 2005;36:934–7. [17] Jennett B, Teasdale G, Braakman R, Minderhoud J, Knilljones R. Predicting outcome in individual patients after severe head-injury. Lancet 1976;1:1031–4. [18] Wilterdink JL, Bendixen B, Adams HP, et al. Effect of prior aspirin use on stroke severity in the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Stroke 2001;32:2836–40. [19] Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5. [20] Banks JL, Marotta CA. Outcomes validity and reliability of the modified Rankin scale: implications for stroke clinical trials — a literature review and synthesis. Stroke 2007;38:1091–6. [21] Vandooren J, Van den Steen PE, Opdenakker G. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade. Crit Rev Biochem Mol 2013;48:222–72. [22] Xi GH, Keep RF, Hoff JT. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 2006;5:53–63. [23] Hoehe MR. Haplotypes and the systematic analysis of genetic variation in genes and genomes. Pharmacogenomics 2003;4:547–70.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
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 outcome: A cohort study in Chinese Han population Jie Yang a,b,1, Sen Lin a,1, Junshan Zhou b, Bo Wu a, Wei Dong a, Hisatomi Arima c, Hua Liu d, Jing Zhang a, Jie Li a, 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 The George Institute for Global Health, Royal Prince Alfred Hospital, University of Sydney, Sydney, NSW 2050, Australia d Department of Neurology, The Third People's Hospital of Mianyang, Luzhou Medical College, Mianyang 621000, China b c
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 20 April 2014 Accepted 12 May 2014 Available online xxxx Keywords: Matrix metalloproteinase 9 Genetic variation Single nucleotide polymorphism Haplotype Intracerebral hemorrhage Outcome
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) outcome in Chinese Han population. Methods: The clinical data and peripheral blood samples from the patients with ICH were collected. The patients were followed up for 3 months, and poor outcome was defined as death or dependency (modified Rankin scale score of 3–6). MassARRAY Analyser was used to genotype the tagger single nucleotide polymorphisms (SNPs) of MMP9 gene. Construction of haplotypes and genetic comparisons were performed by employing PLINK 1.0.7 software. Results: 181 patients with ICH were recruited between September 2009 and October 2010. Information on genetic variations and follow-up assessments were available for 169 (93.4%) patients. Independent patients at 90 days were younger than those who died or dependent (57.82 ± 13.47 vs. 66.99 ± 11.49 years, p b 0.01). In addition, independent patients had lower National Institutes of Health Stroke Scale (NIHSS) score (4[1–6] vs. 13[9–19], p b 0.01). There were no significant associations (all p N 0.05) between MMP9 genetic variations (alleles, genotypes and haplotypes) and ICH outcome after adjustment for conventional risk factors. Conclusions: The genetic variations of MMP9 gene were not significantly associated with ICH outcome at 90 days in 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]. Better management of ICH requires better knowledge on genetic as well as on conventional risk factors of poor prognosis. Brain injury after ICH involves different mechanisms such as physical disruption and mass effect due to hematoma and cerebral edema, and secondary adverse effects of coagulation cascade, hemoglobin breakdown products, and inflammation [4]. Among these, hematoma volume is one of the most important determinants of poor outcome in ICH [5]. Development of perihematomal cerebral edema also leads to subsequent poor clinical outcomes [6,7]. Studies indicated that matrix
⁎ 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 These authors contributed equally to this work.
metalloproteinase 9 (MMP9) levels increased in plasma samples of patients with ICH [8,9]. Increased plasma MMP9 levels were also found to be associated with hematoma growth and perihematomal cerebral edema both in ICH animal [10,11] and in ICH patients [12,13]. Moreover, MMP9 level was predicted by its genetic variations [14]. However, there is no previous report on whether MMP9 genetic variations predict poor prognosis after ICH. We aimed to systematically explore the association between MMP9 genetic variations (alleles, genotypes and haplotypes) and ICH outcome through a larger sample cohort study in Chinese Han population. 2. Materials and methods 2.1. Subjects Our cohort consisted of consecutive Chinese Han patients (n = 181) experiencing a deep location of ICH with hypertension who admitted to West China Hospital or Nanjing First Hospital between September 2009 and October 2010. Clinical diagnosis of ICH was made according to the WHO criteria and was confirmed by brain CT or MRI scans [15]. Other hemorrhagic strokes such as subarachnoid hemorrhage, primary
http://dx.doi.org/10.1016/j.jns.2014.05.021 0022-510X/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
2
J. Yang et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
2.3. Follow-up and outcome
Table 1 Demographic and clinical characteristics of the participants. Characteristics
Death or dependency (n = 74)
Independency (n = 95)
p
Age (year) Male SBP (mmHg) DBP (mmHg) GCS score, median (IQR) NIHSS score, median (IQR) Hypertension Diabetes Hyperlipidemia Stroke Current drinker Current smoker
66.99 ± 11.49 57/77.0% 166.92 ± 25.83 95.16 ± 16.02 13 (8–15) 13 (9–19) 65/87.8% 6/8.1% 2/2.7% 7/9.5% 19/25.7% 18/24.3%
57.82 ± 13.47 68/71.6% 166.99 ± 28.76 100.07 ± 19.26 15 (14–15) 4 (1–6) 80/84.2% 6/6.3% 3/3.2% 6/6.3% 25/26.3% 29/30.5%
b0.01 0.48 0.99 0.08 b0.01 b0.01 0.66 0.77 1.00 0.56 1.00 0.39
SBP, systolic blood pressure; DBP, diastolic blood pressure; GCS, Glasgow Coma Scale; NIHSS, National Institutes of Health Stroke Scale; values are mean ± SD, median (IQR) or n/%.
intraventricular hemorrhage, and ICH due to traumatic, vascular structural abnormalities, antithrombotic therapy, or other known secondary causes were excluded. Deep location of ICH was defined as periventricular white matter, caudate, globus pallidus, putamen, internal capsule, and thalamus [16]. 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 were collected. Detailed information about patient demography (age and gender), conventional risk factors, and stroke severity were collected at baseline. Severity of stroke was evaluated with the Glasgow Coma Scale (GCS) and the National Institutes of Health Stroke Scale (NIHSS) [17,18]. This study was approved by the Ethics Committees of each hospital. Participants were prospectively registered and followed up 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 (SNPs) 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 00.1, r2 N 0.8 and Chinese Han Beijing population [19]. Genotyping was carried out at the Shanghai Benegene Biotechnology Co., LTD (Shanghai, China. http://www. benegene.com.cn/snp/english_site/index.html), using MassARRAY technology platform (Sequenom, San Diego, USA). Haplotypes were identified using PLINK 1.07 (http://pngu.mgh.harvard.edu/purcell/ plink).
The participants were prospectively followed up at 3 months after ICH. The primary outcome was death or dependency at 3 months after ICH. Death was defined as the cumulative all-cause death. Dependency was defined as modified Rankin scale (mRS) score of 3–5 [20].
2.4. Statistical analysis Binary data were described as percentages while continuous data were expressed as the mean ± standard deviation (SD). The Hardy– Weinberg equilibrium was assessed using a χ2 test with 1 degree of freedom. Three genetic models (allelic comparison, dominant and recessive model) were assumed while performing analyses of the distribution of alleles and genotypes between groups. After adjustment for gender, age, and baseline NIHSS score, and controlling for multiple tests with the number of SNPs genotyped, statistical significance of allele, genotypes and haplotypes on ICH outcome was assessed by logistic regression with Plink 1.0.7. A p-value b 0.05 (two-sided) was considered statistically significant.
3. Results The demographic and clinical characteristics of the participants were summarized in Table 1. At the end of 3 months, DNA data and follow-up data were available for 169 (93.4%) patients. Independent patients at 90 days were younger than those dead or dependent (57.82 ± 13.47 vs. 66.99 ± 11.49 years, p b 0.01). In addition, independent patients had higher GCS (15[14, 15] vs. 13[8–15], p b 0.01) and lower NIHSS scores (4[1–6] vs. 13[9–19], p b 0.01). The 6 tagger SNPs (rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577) within MMP9 gene were genotyped in those ICH patients. More than 95.0% participants were genotyped for the 6 tagger SNPs, and 4 haplotypes were successfully identified. Distribution of all genotypes was in the Hardy–Weinberg equilibrium in this cohort (p N 0.05). Table 2 showed the allele frequencies in ICH patients. After adjustment for conventional risk factors (gender, age, and baseline NIHSS score) and multiple tests, there were no significant differences in frequencies between dead or dependent patients and independent ones (all p N 0.05). Table 3 illustrated the genotype frequencies in ICH patients. After adjustment for the same conventional risk factors and multiple tests, there was no significant association between genotypes of MMP9 gene and death or dependency in dominant or recessive models (all p N 0.05). Table 4 demonstrated the haplotype frequencies in ICH patients. Four haplotypes were constructed with the 6 tagger SNPs of MMP9 gene. There was no significant association between the haplotypes and death or dependency after adjustment for the same conventional risk factors and multiple tests (all p N 0.05).
Table 2 Association between alleles of MMP9 and ICH independency at 90 days. SNPs
A1/A2
Death or dependency (n)
Independency (n)
Odds ratio
pa
pb
rs3918241 rs1805088 rs17576 rs3918254 rs3787268 rs17577
A/T T/C A/G T/C A/G A/G
18/124 2/146 41/107 23/125 62/82 19/129
27/149 2/188 47/143 44/146 64/116 29/161
1.77 0.38 1.20 0.53 1.05 1.64
0.30 0.54 0.63 0.18 0.91 0.32
0.99 0.99 0.99 0.99 0.99 0.99
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; A1, allele 1; A2, allele 2. a Adjusted by gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
J. Yang et al. / Journal of the Neurological Sciences xxx (2014) xxx–xxx
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Table 3 Association between genotypes of MMP9 and ICH independency at 90 days. SNPs
Genotypes
Death or dependency (n)
Independency (n)
Comparison models
Odds ratio
pa
pb
rs3918241
AA AT TT CC CT TT AA AG GG CC CT TT AA AG GG AA AG GG
2 14 55 72 2 0 2 37 35 51 23 0 11 40 21 2 15 57
2 23 63 93 2 0 8 31 56 55 36 4 12 40 38 3 23 69
Recessive
AA/(AT + TT)
Dominant Dominant
TT/(AT + AA) CC/(CT + TT)
Recessive Recessive
TT/(CT + CC) AA/(AG + GG)
Dominant Dominant
GG/(AG + AA) CC/(CT + TT)
Recessive Recessive
TT/(CT + CC) AA/(AG + GG)
Dominant Recessive
GG/(AG + AA) AA/(AG + GG)
Dominant
GG/(AG + AA)
18.65 – 1.38 0.38 – & 0.10 – 2.236 0.53 – b0.01 0.62 – 1.40 7.37 – 1.42
0.04 – 0.59 0.54 – & 0.04 – 0.09 0.19 – 1.00 0.50 – 0.52 0.12 – 0.54
0.24 – 0.99 N1 – & 0.24 – 0.99 0.99 – 0.99 0.99 – 0.99 0.72 – 0.99
rs1805088
rs17576
rs3918254
rs3787268
rs17577
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; &, not suitable for statistical analysis due to minor genotype frequency b0.05. a Adjusted for gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
Table 4 Association between haplotypes of MMP9 and ICH independency at 90 days. SNPs
Haplotypes
Death or dependency (%)
Independency (%)
Odds ratio
pa
pb
123456 123456 123456 123456
ACGCGA TCACGG TCGCAG TCGTGG
0.127 0.275 0.444 0.155
0.1534 0.250 0.368 0.229
1.72 1.14 1.04 0.64
0.29 0.73 0.90 0.35
0.99 0.99 0.99 0.99
MMP9, matrix metalloproteinase 9; ICH, intracerebral hemorrhage; SNPs, single nucleotide polymorphisms; 123456, indicate rs3918241, rs1805088, rs17576, rs3918254, rs3787268, and rs17577, respectively. a Adjusted by gender, age, and National Institutes of Health Stroke Scale score. b Statistical adjustments for multiple tests.
4. Discussion We hypothesized that there would be roles of MMP9 genetic variations (alleles, genotypes and haplotypes) on ICH outcome. However, there was no significant association between MMP9 genetic variations and death or dependency at 90 days after ICH onset in the present Chinese Han population. The human MMP9 gene was mapped to chromosome 20q11.2-13.1. Its product is a gelatinase B that fulfills the role of degrading the extracellular matrix both in the vessel and in the brain, and which exacerbate the hematoma growth and cerebral edema after ICH [10–13,21]. The MMP9 gene variations had been proposed to be implicated in poor outcome after ischemic stroke in Caucasian population [8]. However, recovery processes after ischemic stroke and hemorrhagic stroke may be different which led us to explore whether MMP9 gene variations are associated with ICH outcome [22]. To the best of our knowledge, we first systematically explored the association between MMP9 genetic variations and ICH outcomes in a larger (n = 169) sample of Chinese Han population. However, our results indicated that there was no significant association between genetic variations of MMP9 and ICH outcome even after more powerful haplotype analysis (a method of retaining most of the information in a gene and with reducing genotyping requirements) [23]. The current study has several strengths, including the homogeneity sample in the same Chinese Han population. In addition, only patients with deep location of ICH were included because superficial location of ICH maybe often caused by other reasons such as vascular malformation and cerebrovascular amyloid angiopathy. Furthermore, comprehensive analyses of MMP9 genetic variations include the alleles, genotypes and haplotypes, which are more powerful to explore the association between MMP9 genetic variations and ICH outcome [23]. Some limitations should also be noted. First, ICH is one of the complex and polygenic diseases. Although we explored the MMP9 genetic
variations systematically, we might ignore the interaction between MMP9 gene and other candidate genes for ICH outcome. Gene–gene interactions should be considered in future studies. Second, some frequencies of the genetic variants were under 5%, which may lead to insufficient statistical power of our analyses. Furthermore, there was no information on ICH hematoma volume in this registry. So we did not know if the hematoma volume had influenced our results. However, our multivariable analysis included admission NIHSS score as a covariate, and we believe that lack of information on hematoma volume does not completely invalidate the findings of the present analysis. In summary, the MMP9 genetic variations were not significantly associated with ICH outcome in the present Chinese Han population. Future studies should be performed with larger samples and among other ethnic populations, with gene–gene interactions being considered. Conflict of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This study was supported by grants 30900472 and 81100859 from the National Natural Science Foundation of China, grant 2012M511302 from China Postdoctoral Science Foundation, and project 201208008 from Nanjing Health Bureau. We thank Dr. Xiang Liu from West China School of Public Health, Sichuan University for his statistical help. References [1] Krishnamurthi VLF Rita V, 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.
Please cite this article as: Yang J, et al, Genetic variations of MMP9 gene and intracerebral hemorrhage outcome: A cohort study in Chinese Han population, J Neurol Sci (2014), http://dx.doi.org/10.1016/j.jns.2014.05.021
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