Endothelial Nitric Oxide Synthase 3 ( eNOS 3 ) Gene Polymorphisms and Essential Hypertension in Javanese Ethnic Group

Hypertension is still a major public health problem in Indonesia and in several other countries. This disease is caused by multi factorial components involving both environmental and genetic factors. eNOS3 gene is one of the enzymes related to the high prevalence of hypertension. This gene expresses the NOS enzyme which regulates the synthesis of NO. NOS enzyme causes vasodilatation which decreases peripheral resistance and lowers blood pressure. This cross sectional study compared hypertension patients to those with normal blood pressure in the age group of 40-80 years old. The main purpose of this study is to evaluate the influence of eNOS3 gene Glu298Asp allele expression in Javanese ethnic group patients with hypertension. The samples consist of 50 respondents with hypertension and 50 respondents with normotension as control. Data of eNOS3 gene polymorphisms and NO plasma levels from the respondents were analyzed using t-test and chi-square test. Glu298Asp allele genotype variation in eNOS3 gene was detected by PCR-FRLP using primers G894TF and G894TR and the PCR products were cut using Mbol restriction enzymes. Sequencing result of each polymorphism band shows a typical nucleotide sequence compared to the nucleotide sequence of eNOS3 gene in Gen Bank. The results of this study showed no connection between Glu298Asp allele polymorphism in eNOS3 gene with hypertension in Javanese. There was also no relation between eNOS3 gene polymorphisms with high levels of respondents' NO plasma. Average NO plasma level of hypertension patients is 34,53 μmol/L, whereas average NO level of normal blood pressure is 32,5 μmol/L.


INTRODUCTION
explained to all of the patients before they provided formal informed consent.This study with human subjects was Currently hypertension grips around 29,8% of the approved by the regional bioethics committee at the Indonesian population (1).This disease caused by Government of Indonesia.multifactorial components involving both environmental and genetic factors.Clinical and experimental studies Blood Platelet Isolation suggest that more than 90% of the hypertensive patients 10 ml of venous blood was taken from every subject in around the world suffer from essential hypertension (2).
sterile conditions into a tube containing EDTA Many genes involved in blood pressure regulation have potassium salt (11,7 mM) as an anticoagulant (Sarstedt).been screened and recognized as candidates for Plasma and platelets were separated by centrifuging hypertension, such as endothelial nitric oxide synthase (1000 × g) of whole blood for 5 min.Platelets remain on (eNOS) gene.This gene encodes the protein eNOS, the the bottom than resuspension in Tyrode buffer main source of endothelial derived relaxation factor NO containing: 137 mМ NaCl, 12 mМ NaHCO3, 2 mМ KCl, 0,34 under physiological conditions.An association between mМ Na2HPO4, 1 mМ MgCl2, 5,5 mМ glucose, 5 mМ Hepes altered NO metabolism and hypertension has been found (pH 7,3), containing 0,35% bovine serum albumin.Plasma both in animal and clinical studies (3)(4)(5)(6).Because in supernatant collected to measure nitrite concentration endothelial NO availability is regulated at the level of DNA Extraction synthesis, the eNOS gene is hypothesized to be a candidate for essential hypertension and draws Packed cells were lysed with Red Cell Lysis buffer.The considerable attention in many countries (7-8).
solution was centrifuged at 4000rpm for 15min at 4°C to pellet out the nucleated cells i.e.WBCs.Nucleated cells The NOS3 gene is located on chromosome 7q35-36 and were subjected to detergent (10% SDS) and protease consists 26 exons in spans 21 kb.Several studies have been (Proteinase K) treatment in Sodium Chloride-EDTA buffer shown three single nucleotide polymorphisms (SNPs) in and left at 37°C overnight on a shaker.Subsequently the NOS3 gene be associated with hypertension, including proteins were salted out with 5M NaCl.Proteins were G894T, T786C and a VNTR in intron (9-12).The G894T pelleted out by centrifugation at 4000 rpm at room polymorphism, a G to T conversion at nucleotide position temperature for 15 min.DNA was precipitated by ethanol 894 in exon 7, results in a replacement of glutamate by addition to the supernatant.DNA isolated is stored in TE aspartate at codon 298.This variant is not located in any buffer and stored at 4°C for further use.To measure the functional consensus sequence, but computer analysis c o n c e n t r a t i o n a n d p u r i t y o f D N A w e u s e has revealed that the G894T mutation results in a spectrophotometer.conformational change in the eNOS protein from helix to tight turn [14].The Asp variant of this polymorphism is Genotype Determination associated with, coronary diseases (14,15), hypertension A set of primers was designed to amplify a 206 bp fragment (16) and vascular responsiveness to phenylephrine (17).
3'(reverse)].Each PCR was carried out by using genomic Although previous evidence suggests that allelic DNA as the template in final reaction volume of 20 μL polymorphism of endothelial NO-synthase (eNOS) may containing 10 mM Tris chloride pH 8,3, 50 mM KCl, 1,5 mM account for a considerable proportion of the risk of MgCl2, 200 μM each of the four dNTPs, 1 μM each of the development of cardiovascular diseases of complex primers, and 2U Taq DNA polymerase with the following traits (14), the mechanism of the phenotypic expression cycling conditions: 94°C for 45 s, 59°C for 30 s, and 72°C for of pathologic allelic variants of the eNOS gene in blood 45 s for 30 cycles in a PTC 100 (MJ research co.)DNA pressure traits still remains underexplored.In this study, thermal cycler.The PCR fragment digested three hours at we performed a carefully conducted family-based 37°C with the MboI restriction enzyme and then separated association study (90 participants) in Javanese trait in by electrophoresis on 5% agarosa gel and visualized by Indonesia.Our main aim was to estimate the effect of ethidium bromide.polymorphisms in eNOS on essential hypertension eNOS Activity susceptibility in Javanese trait.
For an assay of eNOS activity we used a ELISA based on METHOD Griess method.Nitrite levels were measured using a commercial Griess reaction kit (Cayman Chemicals, Ann

Study Group and Inclusion Criteria
Arbor, MI).The principle of fluorescence of Consecutive hypertensive patients (n=49) admitted in the triazolofluoresceine, which is formed after interaction of State Hospital of Margono Soekarjo, Purwokerto and NO with 4,5-diaminofluoresceine, which is formed from Clinical Private Hospital in Purwokerto was selected.The 4,5-diaminofluoresceine diacetate (DAF-2A) under the criteria for inclusion were: Javanase race and absence of action of intracellular esterase.The wave length of degenerative, systemic and chronic diseases, myocardial excitation/emission was 492/515 nm.The NOS inhibitor revascularization surgery, coronary angioplasty and diphenyliodonium chloride (100 µM) inhibited the previous myocardial infarction.The control group reaction and this confirms the specificity of the NOS consisted of 49 normotensive individuals.Hypertensive activity assay.Enzyme activity was evaluated in units of patients were diagnosed after presenting a constant fluorescence (UF) per min per 106 cells.In mammals systolic arterial pressure, confirmed in three following there are few cell types, which express only one measurements, higher than 140 mmHg and a diastolic isoform of NO synthase.In platelets under normal arterial pressure higher than 90 mmHg.All of the conditions only endothelial NOS is expressed, which procedures, risks and potential benefits were properly ensures that only the activity of this particular isoform was assayed.

Statistical Analysis.
Data were expressed as means ± standard deviation (SD), median (inter quartile range), or absolute number (percentage) when appropriate.The t-test for

RESULTS
result of sequencing is than alignment with prefers data NOS3 nucleotide from gene bank Table 1 summarizes the clinical and laboratorial characteristics of the 100 subjects enrolled in the present study.There were no statistically significant differences in gender, between hypertensive groups and the control group (all P N 0,05).Higher systolic and diastolic blood pressure were found in women with gestational hypertension or with preeclampsia compared with the other groups (both P b 0,05; Table 1).

between Hypertension groups and Normotension (control)
Point mutation in this site make the enzyme cannot work.The gi2315713 groups is reference Sequencing of NOS3 gene from Gen bank The distribution of genotype variation on NOS3 gene from Javanese ethnic presented in Table 2.These data indicated that genotype variation show a great effect on prevalention on hypertension (OR=2,945).Person with hypertension are dominated in genotype GT with close to 60%, but people with normal blood pressure are dominated in genotype GG with the some portion.On the other hand Genotype TT is very rare in both groups.studies have shown a very similar results with 43% decline in patients as compared to controls (20,21).The normal range of NO showed a wide variation in different studies (22)(23)(24)(25).No relationship could be found between nitrite levels and gene polymorphism.This study did not find any correlation between the presence of the eNOS variant and hypertension.This may be because of the small sample size.According to the previous literature, only two studies, one on Caucasians in general (26), and another on the Ukrainian population (23), have shown a correlation between this polymorphism and essential hypertension.

Table 2. Genotype and allele frequencies for NOS3 polymorphisms in both hypertension and normotension group
Other studies have found no such correlation (27,28).Although no statistically significant correlation could be found in the present study, a trend toward a higher frequency of the allele was seen among the patients with conduct a large cohort study so that the nature of any association between essential hypertension and this polymorphism can be tested.If the allele is found to be a Figure 4, shows that there was no significant difference in disease-associated allele, screening of the population for the levels of nitrite between GG and GT genotypes in both individuals at risk might help save lives.If not, we can rule patients (p=0,66) and controls (p=0,46).However, in both out an association of essential hypertension with this the genotypes, levels of nitrite were significantly lower in polymorphism.NO levels showed a significant difference patients than in controls (p<0,01 for ab and p<0,001 for between patients and controls.This suggests that an bb).
estimation of NO levels could be included as a routine lab investigation to screen people at risk and to devise DISCUSSION appropriate individualized therapeutic strategies.However, we stress that the reference value for NO in The participation of females in this study was nearly normal Javanese trait subjects remains to be established.threefold than that of the males.This could be due to the Estimating total NO is rather cumbersome, as it involves social forces in the society where this study was converting nitrate back to nitrite using the enzyme nitrate conducted.Another study done in Padang, Sumatra Barat reductase.However, estimation of nitrite alone using an in Minang ethnic populations, with a larger sample size economic and simple method is a workable alternative.showed the females subjects dominated 78,5% of distribution than male (19).

Figure 2 .
Figure 2. Electroforegram of PCR-RFLP from some samples to

Figure 3 .
Figure 3. Sequencing product of genotypic variation on NOS3 gene Note: Sample 19 and N2 are indicated different genotype.Sample N2 can

Figure 1 . 4 :
Figure 1.Electroforegram of PCR Product of fragment NOS3 gene Note: The 206 bp band is the amplification of fragment gene NOS3

Figure 4 :
Figure 4: Intergenotypic variations in the levels of nitrite in

Table 1 . Clinical and laboratorial characteristics of respondent
cut with MboI but sample 19 cannot cut.GATC is a cutting site of MboI.