The Association Between Lacunar Infarction And Carotid Or Intracranial Artery Atherosclerosis And Notch3 Gene Polymorphisms

Cerebral ischemia stroke is known as the most common disease in neurological clinic. Lacunar infarction(LI) is one important type of cerebral ischemia stroke. Lacunar lesion was defined as a deep subcortical infarction lesion with a diameter of 3-15 mm in the perforating arterial territory. LI is common in older age especially in those who are older than 60 years. It accounts for about a quarter of all types ischemic stroke. It is common in Chinese ischemia stroke patients too. Usually, the pathogenesis of LI is considered as small artery disease(SAD). The pathology mechanism of SAD is lipohyalinosis, arteriolosclosis, reduced cerebral blood flow, vascular endothelial dysfunction, ect. Studies found hypertension was the major pathogenesis of SAD. But recently, the large artery atherosclerosis in lacunar patient is valued more.Notch3 gene is important during vascular development and postnatal arteriogenesis. Notch signalling has been involved in cell-fate decisions and differentiation of epithelial, neuronal, bone, blood, muscle, and, more recently, endothelial cells. Notch3 gene mutations are responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukocephelopathy(CADASIL), whose main characteristic is small vascular degeneration, indicating that Notch3 gene plays an important role in maintaining structural stability and function of vascular smooth muscle cells(VSMC).Gene single nucleotide polymorphisms(SNPs) mean the single nucleotide variation cause the DNA sequence polymorphisms in genome. Many SNPs have been identified in the Notch3 coding sequence, some of them leading to amino acid substitutions too. However, it is not clear whether these polymorphisms affect Notch3 signalling pathway or whether they are involved in cerebrovascular disease and the artery atherosclerosis formation.Studies found that large artery atherosclerosis also was associated with LI. Some genes have affection on the cerebral ischemia stroke too. It is known several single gene diseases such as CADASIL, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukocephelopathy(CARASIL), amyloid artery disease, sickle cell disease could cause LI, and multi-gene interact may increase the risk of the cerebral infarction stroke, such as MTHFR 677 TT, ALOX5 AP 2354AA and Notch3 381TT/TC associated with the increase of cerebral infarction stroke.The patients and the family members would neglect the primary and second prevention on lacunar infarctions because the mild symptoms of LIs. Actually, the recurrence stroke in the LI patients is not so rare. LI also is a risk factor of vascular dementia(VD). If we can make clear the risk factors of LI include carotid or intracranial artery atherosclerosis and Notch3 gene SNPs, we can prevent stroke current or recurrent early. The cost will be decreased due to decrease the incidence of stroke and the disablility caused by stroke.In order to know if carotid or intracranial artery atherosclerosis and Notch3 gene SNPs affect the onset of LI, we enrolled the lacunar patients from our department from March 2010 to December 2011, and analyzed the locations and numbers of LI, their traditional vascular risk factors and the incidences of carotid or intracranial artery arteriosclerosis, and identify the allele frequencies in Notch3 gene, to know the association between the Notch3 gene SNPs, the carotid or intracranial artery arteriosclerosis and LI.Patients, materials and methods:1. Patients:Those who were diagnosed as LI and transient ischemia attack(TIA) were enrolled from March 2010 to December 2011 hospitalized in our department, the lacunar lesions were confirmed by brain CT or MRI scans. Those who have no any cerebral infarctions were regarded as the control subjects.2. Data collection:All patients were performed the brain CT or MRI scans to confirm or exclude the LI. Lacunar lesion was defined as an ischemia lesion in diameter of 3-15 mm in brain imaging. The medical histories of patients include hypertension, DM, hyperlipidemia, heart diseases, smoking, drinking were recorded too. To check the blood pressure, blood glucose and the blood lipid at the first day inhospital, recorded to find undiagnosed hypertension, DM and hyperlipidemia. Carotid and intracranial artery atherosclerosis was assessed by carotid artery ultrasounds, carotid and brain MRA or carotid and brain CTA. Those patients we get the informed consents were analyzed the polymorphisms on Notch3 gene exons 3-6.3. PCR:To identify polymorphisms in Notch3 gene, genomic DNA was obtained from total blood using a Human Blood DNA Kit(Tian Gen). DNA was confirmed by electrophoresis, stored at-20°C prior to genotyping. The Notch3 gene exons 3-6 were amplified by the polymerase chain reaction(PCR) to analyze the SNPs in Notch3 exons 3-6. The primers(designed by gene HUADA company, Bei Jing) for exons 3 and 4 were: forward 5’-GTTTGCTGCTCTGTTTCCCTG-3’ and reverse 5’-GGCACAGTCGTAAGTGAGGT-3’. The PCR conditions consisted of one cycle of 10 min at 95°C, 36 cycles of 30 s at 94°C, 1 min at 65°C, followed by 30 min at 72°C in a Gene Amp PCR system 2400. The PCR product was 656 base pairs. For exons 5 and 6, it is the reverse reaction, the primers were: forward 5’-AGAAAACGGCCACTCACCAG-3’ and reverse 5’-ACACCGATGTCTCAAT GGGG-3’ at an annealing temperature of 55°C. The PCR product was 415 base pairs.4. Gene analysis:Direct sequencing of the PCR products were carried out by the Genemine Company in Chong Qing, China, and GENE TOOLS software was used to identify SNPs. The position of the nucleotide sequence was based on the reference sequence provided by the National Centre for Biotechnology Information(NCBI nucleotide). The Single Nucleotide Polymorphism database of the NCBI was used(www.ncbi.nlm.nih.gov/SNP/) to confirm the SNPs.5. Statistical analyses:The differences in genotype frequencies and other risk factors were analyzed by the χ2 tests, if nessesary, Fisher’s exact test was performed. Mean ages in the two groups and allele frequencies were compared using the independent samples t-test. The association between age, gender, cerebrovascular risk factors, carotid artery atherosclerosis and intracranial artery atherosclerosis with LI stroke were first evaluated in Binary logistic regression model then analyzed in multiple logistic regression. The correlation analysis was done to know the correlation between Notch3 SNPs(with non-stroke as reference) and LI. P were two tailed test, P<0.05 was assumed statistically significant difference. All analysis was performed by SPSS16.0.Results:We enrolled 276 LI patients and 118 controls. The ages in these two groups respectively were 68.9±10.9 and 58.9±10.2 years. The male gender rates are 50.4%(n=139), 44.9%(n=53), respectively. The most common sites of LI lesions located in the basal ganglia region(143cases, 51.8%) and periventricular area(100cases, 36.2%). The main risk factors of LI still were age, hypertension(73.2% and 46.6% in two groups), DM(30.8% and 21.2% in two groups respectively), hyperlipidemia(55.1% and 50.0% respectively). The carotid and intracranial artherosclerosis were not rare in LI patients too. Logistic regression analysis showed that age(OR: 1.803, 95%CI: 1.056-1.112), hypertension(OR: 4.053, 95%CI: 2.421-6.787) and intracranial artery atherosclerosis were significant associated with LI. Intracranial artery atherosclerosis was independently associated with LI in univariable and multivariable analysis even after adjust age and gender.(OR: 4.141; 95%CI: 2.451-6.997 and OR: 3.103; 95%CI: 1.707-5.640respectively). There was no association between heart diseases and LI.There were 135 cases who undertook the Notch3 gene analysis. Those were 105 LI patients and 30 controls. Notch3 gene has many SNPs in exons3-6, but only rs3815188 and rs1043994 have more allelic frequencies in our study. Notch3 gene SNP rs1043994 was associated with LI(correlation analysis P<0.05),there wasn’t relation between rs3815188 and LI. There were not relations between rs1043994, rs3815188 and large artery atherosclerosis.Conclusions:LI was the result of multi risk factors. There was relation between the SNPs rs1043994 and LI. There was no relation between the SNP 3815188 and LI. Intracranial artery arteriosclerosis, age, hypertension were associated with LI. There were not relations between SNP rs1043994, rs3815188 and large artery atherosclerosis. Our data suggest the damaged small vessel in LI was the distant artery or the penetrating artery in brain, SAD was not the only reason for LI, the atherosclerosis and gene were the risk factors of the LI too. Those patients presenting with LI should be included in secondary prevention for large artery atherosclerosis and gene risk. Those patients should screen gene risk and perform angiography.