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The Association Between the Genetic Variants of the NOTCH3 Gene and Ischemic Stroke Risk

Background Ischemic stroke (IS) is a leading cause of disability and death and NOTCH3 as a gene related with cardiac-cerebral vascular disease plays a vital role in IS development. However, the reports about the effect of genetic variants in NOTCH3 gene on IS are still few. Material/Methods In order to explore the association between NOTCH3 polymorphisms and IS, 134 patients with IS and 115 controls were enrolled in this case-control study. Polymerase chain reaction was used to do the genotyping of polymorphisms. The χ2 test was performed to evaluate Hardy-Weinberg equilibrium (HWE) in the control group and calculate odds ratio (OR) with corresponding 95% confidence interval (CI) which represented the association intensity of NOTCH3 gene polymorphisms and IS risk. Results The genotype frequencies in the control group all confirmed to HWE. TT genotype of 381C>T was associated significantly with IS risk (OR=2.441, 95%CI=1.021–5.837). TC, CC mutant genotypes of 1735T>C had higher frequencies in cases than controls and the difference was significant (P=0.013, 0.041); further, its C allele also increased 0.722 times risk in the case group than controls (OR=1.722, 95%CI=1.166–2.541). Conclusions NOTCH3 381C>T and 1735T>C polymorphisms were associated with IS and might be the risk factors for IS development, but not NOTCH3 605C>T polymorphism.


Background
Stroke is caused by cell death owing to abnormal blood supply in brain with high mortality and disability [1][2][3].The symptoms of stroke are sudden weakness, an inability to move or feel, especially one side of the body, aphasia, hearing disorders, and loss of consciousness [4]. It is divided into hemorrhagic and ischemic stroke (IS) mainly, the latter results in lack of blood flow and finally gives rise to losing part of the brain functions [5,6]. IS, a common type, accounts for 85% of stroke deaths and is effected by multiple factors, such as gene, hypertension, tobacco smoking, diabetes [7]. In 1996, the genetic mutations of NOTCH3 were found to be associated with stroke for the first time [8].
NOTCH reports play key roles in regulating nervous system development and facilitating neural stem cells renewal and proliferation [9,10]. It is encoded by NOTCH gene including four members [11]. The expression of defects and inactivation in NOTCH result in severe nervous and cardiovascular system diseases, sometimes they are fatal [12][13][14]. NOTCH3 is a member of the NOTCH family and can accelerate cell proliferation and gliogenesis [15]. It is also reported to participate in brain development and some tumors occurrence [16,17]. The mutations of NOTCH3 have been identified and cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [18,19]. Similarly, mutations in NOTCH3 gene are found to be associated with Alzheimer's disease in a Turkish family [20]. Further, usually patients with the clinical symptoms of CADASIL present with IS. However, so far, only a few reports explain the relationship between NOTCH3 gene polymorphisms and IS risk in several populations; the etiology of IS is also unclear.
In the current study, we selected 3 polymorphisms in NOTCH3 (381C>T, 1735T>C, 605C>T) to examine the association with IS susceptibility in 134 patients with IS and 115 control subjects who came from the Chinese Han population. We hoped that it provided evidence for explaining the pathology and etiology of IS.

Study subjects
In the case-control study, 134 patients with IS were selected from the neurology department of People's Hospital of Liaocheng diagnosed by clinical pathology in April 2013 to October 2014 as the case group. The patients were aged between 35 and 74 years, and 61.9% of the patients were male. A total of 115 control subjects were frequency-matched with cases for age and sex. The control group comprised patients with mild symptoms from ophthalmology, otorhinolaryngologic, and digestive departments of the same hospital, as well as community residents, who had not had a stroke. The age range was from 33 to 75 years, with an average age of 59.26±9.04. This study design was supported by the Research Ethics Committee of People's Hospital of Liaocheng. All subjects were Chinese Han population without a blood relationship.
We collected the detailed clinical information of every subject after the patient or family member signed the written consent form. Some indexes were examined, such as smoking, alcohol consumption, body mass index, hypertension, hyperglycemia, diabetes. A person who smokes 1 or more cigarettes a day, on average, for at least half a year is a smoker. A person is a drinker who drinks >1 time a week (for men) or ³1 time a week (for women). Average blood pressure through measuring 3 times ³140/90 mm Hg or taking antihypertensive drugs is defined as hypertension. Fasting blood glucose (FBG) level ³6.1 mmol/L is defined as hyperglycemia. FBG level ³7.8 mmol/L, or 2 hours after taking oral glucose ³11.1 mmol/L, is diagnosed as diabetes.

DNA extraction and PCR primer design
From every selected person, 2 mL fasting venous blood was collected using a vacuum collective tube with EDTA anticoagulant. Then genome DNA was extracted by conventional chloroform/ isoamyl alcohol method and finally stored at -20°C refrigerator.

Polymorphisms
Primer sequences Position Length (bp)

CLINICAL RESEARCH
PCR primers were designed in Primer 5.0 software based on GeneBank and the detailed primer sequences were listed in Table 1.

The genotyping of NOTCH3 gene polymorphisms
Polymerase chain reaction (PCR) was used to conduct the genotyping. PCR reaction solution was a total of 20 μL, containing 0.5 μL genome DNA template (20 ng/μL), 1 μL forward and reverse primers (each 0.5 μL, 10 μmol/L), and 10 μL PCR Mix, and finally added to 8.5 μL deionized water. Subsequently PCR system was performed according to the following steps, firstly initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30s, annealing at (65°C for 381 C>T and 1735 T>C, 60°C for 605 C>T) for 30s, extension at 72°C for 1 min and final extension at 72°C for 8 min.
The PCR products were checked in 2% agarose gel electrophores. Sequencing was performed on an ABI 3130 Genetic Analyser after the samples were purified.

Data analysis
All data were represented by c _ ±s or%. The genotype distributions of gene polymorphisms in the control group was checked to make sure it was consistent with Hardy-Weinberg equilibrium (HWE) by c 2 test which was also used to calculate odds ratio (OR) and 95% CI evaluating the association intensity between NOTCH3 gene polymorphisms and IS susceptibility. Statistical analysis was conducted in SPSS 18.0 software and P<0.05 was considered as significant difference.

The characteristics of all subjects
There was no significant difference in cases and controls based on age and gender (P>0.05). As was shown in Table 2, we saw that BMI and alcohol consumption were not the direct risk factors, however, the number of smoker, suffering from hypertension, hyperglycemia, diabetes was obviously large in cases compared with controls and they might be associated with the development of IS (P<0.05).

The comparison of genotype distribution in NOTCH3 gene polymorphisms between the cases and controls
The results were shown in Table 3, TT genotype frequency of 381C>T was higher in the case group than the control group and might be associated with IS risk (OR=2.441, 95%CI=1.021-5.837). Similarly, the polymorphism 1735T>C was also an independent risk factor for IS. TC, CC genotypes had higher frequency in cases compared with control subjects

Discussion
Stroke is the second leading cause of death and disability and occurs mostly in the middle-aged population, but onset age is inclined to be young nowadays [21]. Most strokes are IS [22]. Multiple elements participate in the development and progression of IS, such as genetic variant, hypertension, high cholesterol, and diabetes. In addition, the people exposed to external factors suffer from IS, so the possible result is abnormal expression of relative genes caused by these factors. To explain the pathogenesis of IS, scholars study the effect of gene mutations on IS occurrence.  [25]. In addition, COX, MMP, CCM, and IL relative genes are identified to involve in the pathology of IS. However, the cause of IS is still not completely clear.  3913 verify the association in Caucasians, the result is certain that the polymorphisms of NOTCH3 gene may lessen the risk of IS [28]. The genetic variants of NOTCH3 gene are also usually detected in patients with acute IS in Korea, which indicates NOTCH3 polymorphisms can influence IS [29].
In the current study, subjects were from the Chinese Han population, and 3 polymorphisms of NOTCH3 gene were selected to explore the roles in IS. The homozygous mutant genotype frequency of 381C>T in cases was 2.441 times more than in the control group. The mutant genotypes and allele of 1735T>C in frequency had the significant differences between the 2 groups. These 2 polymorphisms each showed the relevance with IS development in study population, but 605C>T might not be an independent factor in IS.

Conclusions
The accuracy of our results is restricted by some conditions. The study subjects only include the Han population in Shandong Province, China, and the sample size is small. However, we explain the role of only a single polymorphism in IS; the interactions are not considered. Therefore, in exploring the precise pathogenesis of IS, we still have a lot of work to do. We need well-designed studies with sufficient sample size and environmental factors to be performed in the future.