With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases, genetically associated with an autosomal dominant form of hypertension. Argumentative consequences have lately arisen on the association of specific single nucleotide polymorphisms of
Essential hypertension, as a significant health risk factor leading to renal and cardiovascular disorders, is a common disorder developed by unhealthy lifestyle habits and heritable elements. The genetic influence or heritability estimation on blood pressure (BP) variation displays remarkable range (30 to 50%) according to Timberlake et al. [
Previous prospective and case control studies have shown that hypertension progression is an independent predictor of T2DM [
The serine-threonine kinase with-no-lysine (K) 4 gene (MIM # 601844) is a hot locus for blood pressure that is on 17th chromosome [
Considering a strong relation between hypertension and diabetes in this study, our major target aimed at hypertension as well as T2DM to specify role of the gene underlying the disease to modify the prognostication of those at risk and also administer further antihypertensive treatments. The current research principal objective was to explore the relation of WNK4 gene Ala589Ser polymorphism between EHT and T2DM in Malaysian subjects. Several screening tests resulted in the recognition of human-specific variant Ala589Ser in WNK4 exon 8. This polymorphism (Ala589Ser) was targeted to be conducted on Malaysian population through evolutionary genetic analysis and to explore its relation with BP and FBS development as well.
Ethical approval was acquired from Seremban hospital as well as Universiti Putra Malaysia with reference number [UPM.FPSK.PADS/T7-MJETIKAPer/F01-JSB-Mac]. All participants were asked to fill in informed consent questionnaires. In the current study, 320 Malaysian participants were interviewed. The blood pressure was measured on the right arm (three times) and averaged after resting (5 min intervals) by digital Sphygmomanometer on the basis of inclusion criteria [SBP above 140 mmHg and DBP above 90 mmHg (for patients)]. Participants’ height and weight were taken to measure BMI utilizing the formula [weight (kg)/height (m2)]. All participants had biochemical analyzing test using plasma electrolytes.
In the study, the buccal and blood cells were collected from cases (hypertensive patients) and controls, respectively. The blood was kept in ethylene diamine tetra acetic acid (EDTA) tube and stored at 4°C for a maximum of three days before utilizing. The DNA was applied for amplification after extracting it from buccal and blood cell samples using Qiagen kit (Germany); then it was stored at −20°C for later usage. DNA was qualified right after all primers were optimized by PCR method. By utilizing the Nanodrop in two optical density (OD) wavelengths (260 nm and 280 nm), the extracted DNA concentration was examined.
In this section, peripheral venous blood samples were collected after keeping an overnight fast by control subjects. Moreover, lipid profiles [including TG, LDL, total cholesterol (TCH), and high-density protein (HDL)] were determined. We also measured FBS with standard laboratory techniques; however, the diabetic glucose levels were taken from the medical history. It was noticeable that we had referred to the hospital patients’ documents to assess biochemical information for cases.
Genomic DNA was amplified by multiplex-PCR reactions. The examined polymorphism of WNK4 (Ala589Ser) was identified using PCR based RFLP. PCRs were carried out with forward primer 5′-TGGAAACCCATTTTCCCCTGG-3′ and reverse primer 5′-AGGTGGTGAGGCCTAGAAAGT-3′ at the specific temperatures. Each reaction was composed of 6x master mix (which embodies DNA polymerase, MgCl2, dNTPs, and reaction buffers), 0.6
The enzyme
The statistical analysis in present study was conducted by statistical package for the social science (SPSS version 22). Utilizing two-tailed Student’s
In the study, 320 volunteers, divided into hypertensive (163) and normotensive (157), participated for screening Ala589Ser polymorphism in WNK4. The clinical characteristics mean and standard deviation (SD) were clearly indicated in Table
Baseline characteristics of the subjects ±.
Parameter | Hypertensive subjects | Normotensive subjects |
|
---|---|---|---|
Gender (male/female) | (90/74) | (73/83) | NS |
Age (year) |
|
|
<0.0001 |
SBP (mmHg) |
|
|
<0.0001 |
DBP (mmHg) |
|
|
<0.0001 |
BMI chol (mmol/L) |
|
|
<0.0001 |
FBS chol (mmol/L) |
|
|
<0.0001 |
T-chol (mmol/L) |
|
|
NS |
LDL-chol (mmol/L) |
|
|
<0.0001 |
HDL-chol (mmol/L) |
|
|
NS |
TG (mmol/L) |
|
|
0.0001 |
Diabetes (yes/no) |
|
|
0.000 |
Family history (yes/no) |
|
|
0.000 |
Smoking habits (yes/no) |
|
|
0.008 |
Alcohol drinking (yes/no) |
|
|
0.000 |
Data are mean ± SD unless otherwise specified.
Multiple PCRs were used to determine the absence and presence genotype of Ala589Ser of WNK4 gene in samples. Before doing main RCR, clarity of the bands was checked by optimization of the method. Gene was set up separately with internal control to find out the sharp band. Each gel illustrated the optimization on one gene with four different samples. PCR products were run on 2% gel with four different samples; 50 bp ladder was used as a DNA marker. After PCR product digestion by
Detection of Ala589Ser polymorphism of WNK4 gene by PCR-RFLP utilizing 2.0% agarose gel electrophoresis. PCR products were digested by AlwNI. Wild type was cut (into 180 and 112 bp), while variant type remained uncut. Homozygous genotypes are indicated as GG genotypes, heterozygous genotypes are determined as GT genotypes, and TT genotypes show mutant genotypes. M represented a 50 bp DNA ladder plus (Bioline).
Table
Genotype and allelic frequency distribution between hypertensive and normotensive subjects.
Hypertensive subjects | Normotensive subjects |
|
|
---|---|---|---|
Genotype | |||
GG | 95 (58.3) | 109 (69.4) | 0.005 |
TG | 57 (35.0) | 48 (30.6) | |
TT | 11 (6.7) | 0 (0.0) | |
|
|||
Allele frequency | |||
G | 247 (75.8) | 266 (84.7) | 0.015 |
T | 79 (24.2) | 48 (15.3) | |
|
|||
Post hoc test |
|
(95% confidence interval) | |
|
|||
TT versus TG | 0.004 | 0.15–0.76 | |
TT versus GG | 0.001 | 0.23–0.83 | |
TG versus GG | 0.193 | −0.19–0.4 |
In Table
To specify genotype-clinical characteristics correlations, clinical characteristics of patients between genotypes of Ala589Ser polymorphism were contrasted (Table
Genotype-clinical characteristics correlations in patients.
Variable | GG | GT | TT |
|
---|---|---|---|---|
Gender (male/female) | 52 (54.7%)/43 (45.3%) | 31 (54.4%)/26 (45.6%) | 7 (63.6%)/4 (36.4%) | NS |
Age (year) | 55.63 ± 10.21 | 55.83 ± 10.73 | 60.27 ± 12.49 | NS |
SBP (mm/Hg) | 134.97 ± 21.50 | 135.99 ± 22.02 | 152.73 ± 11.69 | NS |
DBP (mm/Hg) | 80.25 ± 9.81 | 82.87 ± 11.22 | 82.91 ± 10.96 | 0.041 |
BMI (cm/kg) | 25.83 ± 4.93 | 26.24 ± 3.92 | 25.87 ± 4.10 | NS |
FBS (m/mole) | 5.51 ± 1.09 | 5.87 ± 1.31 | 5.87 ± 1.01 | NS |
TCH (m/mole) | 4.87 ± 1.26 | 4.64 ± 0.96 | 4.75 ± 0.72 | NS |
LDL (m/mole) | 3.01 ± 1.13 | 2.82 ± 0.95 | 2.91 ± 0.94 | NS |
HDL (m/mole) | 1.30 ± 0.46 | 1.21 ± 0.36 | 1.34 ± 0.35 | NS |
TG (m/mole) | 1.39 ± 0.56 | 1.38 ± 0.56 | 1.34 ± 0.51 | NS |
Diabetes (no/yes) | 46 (48.4%)/49 (51.6%) | 22 (38.6%)/35 (61.4%) | 7 (77%)/4 (44%) | NS |
Family history (no/yes) | 42 (44.7%)/52 (55.3%) | 23 (40.4%)/34 (50.6%) | 8 (72.7%)/3 (27.3%) | NS |
Smoking habit (no/yes) | 77 (81.1%)/18 (18.9%) | 48 (84.2%)/9 (15.8%) | 8 (72.7%)/3 (27.3%) | 0.045 |
Alcohol consumption (no/yes) | 86 (90.5%)/9 (9.5%) | 44 (77.2%)/13 (22.8%) | 10 (90.9%)/1 (9.1%) | NS |
Data are presented as mean ± SD;
In order to confirm the genotyping results, random samples were used and repeated with the same PCR conditions. To receive a final confirmation of the nucleotide sequence, purified PCR products were sent to Research Biolabs Malaysia. The sequencing results were aligned with the gene sequence from the NCBI-GenBank by the MEGA4 software [
We targeted Ala589Ser polymorphism of WNK4 as a candidate gene in hypertension to specify potential association affecting high blood pressure determination. We specified a human-specific polymorphic Ala589Ser in WNK4 exon 8. In spite of the fact that the relevant research on Ala589Ser polymorphism variation of WNK4 gene was rather basic, close relation of WNK4 gene between cases and controls was recognized well recently [
Although some researchers as Lu in china and Lv in Uyghur ethnicity did not observe significant association of Ala589Ser for genotype and allelic frequency [
Guo et al. (2014) also pooled five studies in their meta-analysis; they managed to illustrate significant association between Ala589Ser of WNK4 and hypertension using both dominant genetic (
WNK4 is expressed mainly in collecting ducts and distal convoluted tubules of the kidney and colon. Ala589Ser polymorphism of WNK4 is mapped in
The present study perhaps is the first effort to specify the relation between WNK4 gene Ala589Ser polymorphism and EHT in Malaysia. Significant association of Ala589Ser polymorphism, WNK4 gene, and hypertension has been specified in Malaysian genotype. Close association between TT genotypes/T allele of WNK4 gene and EHT has been demonstrated in contrast to controls in Malaysia (
Considering the fact that 88 individuals out of 163 hypertension cases were diagnosed with T2DM based on their hospital records, a strong relation was established between hypertension with T2DM and Ala589Ser (
Genotypes and allele frequencies distribution of gene polymorphisms between two patient groups and control subjects.
EHT |
EHT+T2DM |
Control |
|
---|---|---|---|
Genotype and allele frequency | |||
GG | 46 (61.3) | 49 (55.7) | 109 (69.4) |
GT | 22 (29.3) | 35 (39.8) | 48 (30.6) |
TT | 7 (9.3) | 4 (4.5) | 0 (0) |
G | 114 (76) | 133 (75.6) | 266 (84.7) |
T | 36 (24) | 43 (24.4) | 48 (15.3) |
|
0.001 |
0.004 |
|
Odds ratio (95% CI) | 571 (252–928) | 0.558 (0.352–885) | |
|
|||
Post hoc test | |||
|
|||
TT versus GT | 0.000 | 0.018 | |
TT versus GG | 0.000 | 0.004 | |
GT versus GG | 0.790 | 0.083 |
Significant differences in age, SBP, DBP, BMI, FBS, LDL, and TG were found between hypertensive and normotensive subjects (
In summary, we analyzed Ala589Ser polymorphism of WNK4 gene. The Ala589Ser polymorphism displayed significant association between genotype/allele frequency and hypertension; this polymorphism has a potential effect on the clinical characteristics profile of the subjects. According to Sun et al., different hormones might have effect on the WNK4 gene expression in the kidneys. Increasing susceptibility in essential hypertension may be significantly influenced by the Ala589Ser polymorphism as a missense mutation [
The findings of the present research indicate that TT genotypes/T allele of WNK4 gene produce a close relationship with EHT and T2DM. The current research must be interpreted within its context restrictions. The proof of the relationship between T/G variant of WNK4 gene and EHT at the gene level has been provided only; nevertheless, the function and mechanism of T variant have not been addressed. Second, in contrast to cases, the control subjects are relatively young but not age matched; however, more relative studies with larger population concerning other candidate gene polymorphisms or T/G polymorphism of WNK4 gene in relation to cardiovascular disorder are suggested.
The authors declare that they have no competing interests.
Nooshin Ghodsian conceived the study and participated in the experimental design, data acquisition and analysis, and interpretation of results and drafted the paper. Nooshin Ghodsian, Patimah Ismail, Salma Ahmadloo, Farzad Heidari, Polin Haghvirdizadeh, Sima Ataollahi Eshkoor, and Ali Etemad interpreted the results and critically reviewed the study for important intellectual content. All authors approved the final version of the paper.
The authors fully acknowledge the cooperation of the volunteers and all participants who generate their generous support. They are also grateful to Mr. Mohammad Akhlaghi who edited this paper grammatically and in that regard improved the paper significantly.