| Stroke is a sudden onset of brain blood circulation disorders, which lead to transient or permanent brain dysfunction clinical symptoms and signs. Ischemic stroke accounts for 80-90% of the total number of patients with stroke. The morbidity and mortality is high, which lead to serious harm to human health and enormous burden of people’s lives. There are many risk factors for ischemic stroke, including diabetes, obesity, high blood pressure, blood viscosity, heart disease. Many previous studies have shown that serum uric acid usually associated with risk factors for atherosclerosis and metabolic disorders and elevated serum uric acid related to the morbidity and mortality of ischemic stroke. However, some scholars have contrary experimental findings. They found that ischemic stroke, caused by a series of metabolic cascade of events, produce large amounts of free radicals and result to oxidative damage to the central nervous system. Uric acid, as a free radical scavenging, may play a protective role in ischemic stroke. Through large-scale clinical studies, they have shown that uric acid is a good prognostic factor for ischemic stroke. Obviously, there is a dispute on the correlation between uric acid and ischemic stroke. There are many etiology of stroke, such as large artery atherosclerosis(LAA), small-artery occlusion lacunar(SAA), cardioembolism(CE) and stroke of other demonstrated etiology(SOE). LAA and SAA type is a major cause of ischemic stroke, and both related to atherosclerotic vascular wall. Therefore, LAA and SAA stroke can be combine into non-cardioembolic ischemic stroke(NCIS). We intend to study the relationship between serum uric acid and non-cardioembolic ischemic stroke. Choosing non-cardioembolic ischemic stroke for the study is conducive. Subtype analysis based on the specific pathophysiology pathways will increase the homogeneity of the studied traits. Therefor, the use of genotyping strategy is more conducive to investigate correlation between uric acid and ischemic stroke.With the advances of molecular biology and neuroscience, it is recognized that the incidence of stroke were associated with the expression and regulation of some genes. Therefore, to prevent and treat the disease by gene regulation has become a hot research. Genetic research does not only provide seeking clues to the etiology of ischemic stroke, but also provide a theoretical basis for prevention, diagnosis, treatment and prognosis of ischemic stroke. Despite there is a dispute on the correlation between uric acid and ischemic stroke, it was recognized by more and more scholars that the uric acid have involved in the onset, development and prognosis of ischemic stroke. Nowadays, the impact of Facilitated Glucose Transporter 9(GLUT-9) gene on serum uric acid levels also becomes the consensus. GLUT-9 gene is located on 4p15.3-p16, with a total length of 214 025bp. It include a non-coding regions exon and 13 coding exons, which encoding glucose transporter 9 (GLUT9) and involved in the metabolism of glucose, fructose and uric acid, etc. Previous studies have demonstrated that GLUT-9 gene mutation can lead to loss function of GLUT9, which will lead to decline of uric acid absorption in the kidneys and cause low serum uric acid. Genome-wide scan analysis found that genetic variation of GLUT-9 gene were associated with serum uric acid levels. This correlation is mapped to the single nucleotide polymorphism(SNP) in a non-coding region, located in the the 5’-untranslated region and intron 3 to 7. There are many studies have shown that GLUT-9 gene promoter SNPs are related to hyperuricemia, gout, diabetes and coronary heart disease. However, to our knowledge, there is not research on GLUT-9 gene promoter SNP and ischemic stroke. To further explore the relationship between GLUT-9 gene SNP and serum uric acid and ischemic stroke, our study using bioinformatics software to predict core promoter region and transcription factor binding sites of GLUT-9 gene. We conduct genetic screening for non-cardioembolic ischemic stroke patients, hoping to find the relationship between gene variants and serum uric acid and clinical features in patients with ischemic stroke.Part I The study of the correlation between serum uric acid concentrations and acute non-cardioembolic ischemic strokeContents and methodsPatients with documented definite LAA and SAA stroke according to the TOAST classification (combined into non-cardioembolic ischemic stroke), older than 18 years, and visiting the second affiliated hospital of Guangzhou Medical University from January 2012 to April 2014 were enrolled in the study. Patients with established transient ischemic attack(TIA), cerebral hemorrhage, brain tumors, brain trauma, severe kidney and liver diseases, leukemia, multiple myeloma, polycythemia vera, autoimmune diseases involving the kidney or without complete clinical data were excluded from the study. The final study population was 516 patients. Progress stroke diagnostic criteria:(1) neurological deficit symptoms and signs progressive increase in one week after the onset, (2) The National Institutes of Health Stroke Scale (NIHSS) score decreased by 2 points or more, (3) exclusion of secondary brain hemorrhage. All patients had given informed consent to participate in the study. Permission for the study was obtained by the local ethics committees.At baseline, demographic data (age, sex), history of conventional vascular risk factors (hypertension, diabetes mellitus, dyslipidemia, ischemic heart disease, stroke, transient ischemic attack, smoking habit and alcohol abuse) were obtained. All blood samples were obtained at the first day of admission. Uricase-peroxidase method has been adopted as a determination of serum uric acid(UA) concentration. All patients received treatment according to the current guidelines. The NIHSS score was assessed on admission. The modified Rankin Scale (mRS) score was assessed at discharge. A score of greater than 2 on the mRS was used to define a poor functional outcome at discharge. TOAST classification was classified according to MR, DSA, echocardiography, carotid ultrasound examination results and so on. DWI infarct volumes (DIV) were measured using 32-bit OsiriX imaging software.Descriptive and frequency statistical analyses were obtained and comparisons were performed with SPSS for Windows, version 13.0. Normally distributed continuous variables were presented as mean±tandard deviations and statistical significance for intergroup differences were assessed by independent samples t test or one-way ANOVA. Not normally distributed continuous variables were expressed as median (inter quartile range) (M, IQR) and statistical significance in different groups were assessed by Kruskal-Wallis test. Categorical variables were expressed as frequency (percentage) and statistical significance for intergroup differences were analyzed with R×C Chi-square test. The strength of associations between UA and stroke outcome were assessed by means of logistic regression analysis. This association was first tested univariately. Multivariate analysis was performed by binary logistic regression analysis, which allows adjustment for all potential confounding factors. A probability value<0.05 was considered statistically significant.Results516 cases of LAA and SAA ischemic stroke patients (combined non-cardioembolic ischemic stroke group) were took part in this study. The average age of enrolled patients was (66.252±12.850) years old,308 cases were male (59.7%) and 208 cases (40.3%) were female. Among the 516 study patients, median UA was 336.5 μ mol/L (interquartile range,131.8 μmol/L; full range,103-658 μmol/L). Serum uric acid of non-cardioembolic ischemic stroke was positively skewed distribution. There were 157 (30.4%) LAA patients and 359 (69.6%) SAA patients.The patients were divided into quintile groups depending on serum SU levels: first quintile,259 μmol/L and below; second quintile,259 μmol/L through 310 μmol/L; third quintile,310 μmol/L through 366 μmol/L; fourth quintile,366 μmol/L 425 μmol/L; fifth quintile, above 425 μmol/L. Those with lower UA levels were more likely to be female and have poor outcome (p<0.05). However, the proportion of NIHSS≥12 scores and progressive stroke difference between the five groups has no statistical difference (p>0.05).The cerebral infarction volume of low serum uric acid levels (≤259 μmol/L) group and high serum uric acid levels (>259 μmol/L) group were [1.512 (7.894)] cm3 and [1.018 (3.363)] cm3, respectively. There was statistical difference between two groups (Z=2.032, p=0.042)In univariate analysis, age (odds ratio(OR):1.028,95% confidence interval(CI): 1.013～1.043;p<0.001), history of stroke (OR:2.112,95% CI:1.349-3.304; p=0.001), NIHSS scores≥12 (OR:24.497,95% CI:7.370-81.419; p=0.001) and UA≤259 μmol/L (OR:2.091,95% CI:1.346-3.246; p=0.001) were correlated with poor outcome. In multivariate analyses, age (OR:1.029,95%CI:1.012-1.046; p=0.001), history of stroke (OR:1.796,95% CI:1.097-2.942; p=0.020), NIHSS scores≥12 (OR:24.485,95% CI:7.168-83.634;p<0.001) and UA≤259 μmol/L (OR:2.069,95% CI:1.282～3.338;p=0.003)were the independent predictive factors for the poor clinical outcome in patients with non-cardioembolic ischemic stroke after adjusting for gender, diabetes mellitus, dyslipidemia, ischemic heart disease, and so on.Conclusion1. Acute phase serum uric acid of non-cardioembolic ischemic stroke patients is a wasting antioxidant. Acute phase serum uric acid is associated with infarct volume and can be used as a index for assessing stroke lesion severity early.2. UA≤259 μmol/L is independent risk factor of short-term outcome in patients with acute non-cardioembolic ischemic stroke. Lower serum uric acid level may be unfavorable to the short-term prognosis of acute non-cardioembolic ischemic stroke. The relationship between uric acid and stroke needs further research.3. Admission serum uric acid level was not linked to hospital illness severity and progressive stroke.Part II The study of the correlation between the GLUT-9 gene promoter region single nucleotide polymorphisms and non-cardioembolic ischemic strokeContents and methodsPatients with documented definite LAA and SAA stroke according to the TOAST classification(combined non-cardioembolic ischemic stroke), older than 18 years, and visiting the second affiliated hospital of Guangzhou Medical University from January 2012 to April 2014 were enrolled in the study. Patients with established transient ischemic attack (TIA), cerebral hemorrhage, brain tumors, brain trauma, severe kidney and liver diseases, leukemia, multiple myeloma, polycythemia vera, autoimmune diseases involving the kidney or without complete clinical data were excluded from the study. The final study population was 86 patients. All patients had given informed consent to participate in the study. Permission for the study was obtained by the local ethics committees. The modified Rankin Scale (mRS) score was assessed at discharge. A score of greater than 2 on the mRS was used to define a poor functional outcome at discharge. Serum uric acid (UA) were measured by using the automatic biochemical analyzer (LX20, Beckman, U.S.A.). Peripheral blood DNA was extracted from 86 patients with non-cardioembolic ischemic stroke, and then GLUT-9 promoter SNP sites were screened by direct sequencing subsequently. Bioinformatics software were used to predict the core region of the promoter and the potential transcriptional regulatory elements. Descriptive and frequency statistical analyses were obtained and comparisons were performed with SPSS for Windows, version 13.0. A probability value<0.05 was considered statistically significant.ResultsBioinfomatic analysis showed that the core promoter of human GLUT-9 gene may be located in-550 to-503. Results showed that 11 SNP were found through screening the non-cardioembolic ischemic stroke patients. These SNPs located in the-2030～-88 region and mostly in the vicinity of the first exon. SNPs found in this study led to the disappearance of a large amount of transcription factors binding sites and the appearance of new transcription factors binding sites. The Serum UA levels and cerebral infarction volume diferences between each genotype group was not statistically significant (p>0.05). No significant correlation was found between different genotypes of the 11 SNPs and prognosis of non-cardioembolic ischemic stroke (p>0.05).Conclusion1. The core promoter of human GLUT-9 gene may be located in -550 to -503.2. SNP variation in the promoter of GLUT-9 gene led to the disappearance of a large amount of transcription factors binding sites and the appearance of new transcription factors binding sites, which may significantly influence the regulation of gene expression.3. SNP variations in the promoter of GLUT-9 gene are not related with the concentration of serum UA level, cerebral infarction volume and prognosis of non-cardioembolic ischemic stroke patients. |
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