| Objective:Hypertension is the most prevalent risk factor for cardiovascular morbidity and mortality worldwide. Indeed, hypertension affects> 18.8% of the adult population in China. According to the data of China National Nutrition and Health Survey in 2002, there are 160 million hypertensive patients in China and most of them (116 million) live in rural. The prevention of hypertension has been an important public health issue of rural area in the underdeveloped regions of China. Hypertension is a complex disease resulted from the interaction of the cumulative effect of multiple genetic and environmental factors and genetic factors play an important role in it. Along with the achievement of human genome project and human haplotype map project, the genetic study of complex disease get into a new era-genome-wide association study (GWAT). Nevertheless, there may still be a need for candidate gene approaches, especially when looking at single-nucleotide polymorphisms (SNPs) with low allele frequencies and rare mutations, especially in special ethnic population. By systemically screening SNPS and rare mutation in coding and regulating region of candidate gene and study the relationship between these variations and hypertension, it is possibal to find variations are related to diseases. This will play an essential role in searching the genetic mechanisms of some complex polygenic diseases. A region on chromosome 18q21-22 has been identified in a number of genome-wide scans on hypertension, therefore making the locus a suitable candidate for harboring gene/genes involved in blood pressure (BP) regulation. The 18q21-22 locus harbors an important BP-regulating gene (the neural precursor cell expressed developmentally down-regulated 4-like [NEDD4L] gene). NEDD4L regulates BP through its ubiquitinating effects on the epithelial sodium channel (ENaC) which is a critical component of the pathway maintaining salt and water balance at the luminal cell membrane in the renal collecting ducts, resulting in reduction of the number of ENaC present on the plasma membrane. Thus, NEDD4L is a candidate gene for hypertension, both functionally and genetically. Epidemiological data indicates that the prevalence of hypertension varies greatly between different ethnic populations. Kazakh, a nomad population which dwells north of Xinjiang in northwest China and in which 99% are herdsman, is characterized by a higher prevalence of hypertension and higher BP levels compared to other ethnic populations residing in the same area. Moreover, very few Kazakh marry people of other ethnicities because the unique customs, cultural background, and food habits are different from other ethnicities. Therefore, Kazakh is a relatively isolated population with a pure genetic background and is an ideal population to study genetic mechanisms of complex diseases, such as hypertension. However, there are no reports regarding the relationship between genetic variations in the human NEDD4L gene and essential hypertension in Kazakh. The purpose of the current study was to investigate the prevalence of hypertension in Kazakh, and the relationship between the variation of NEDD4L and essential hypertension in Kazakh from the pasture area of Xinjiang Fukang.Methods:(1) A population-based, cross-sectional study in a Kazakh general population in the pasture area of Xinjiang Fukang was performed which included 1083 participants (response rate 95.5%) aged 30-70 years and with no mixed marriages within the past three generations by cluster sampling. In this study, a multi-stage sampling method was used to select a representative sample of the population. In addition to performing routine blood testing that included lipid profiles, blood electrolytes, glucose levels, and /urine electrolytes, anthropometric measurements were obtained and a questionnaire was completed.(2) All the sequence variants located promoter and exon regions of NEDD4L were identified by directly sequencing of PCR product. Blood samples were obtained from 94 (47 males and 47 females)hypertensive patients, which were randomly chosen from the hypertensive group of the study population, and genomic DNA was isolated from peripheral blood leukocytes using a PAXgene Blood DNA kit (PreAnalytiX). All exons with their flanking sequences and approximately 500 bp of the upstream region of the promoter were directly sequenced by an ABI 3130X1 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using 35 sets of primers. The obtained sequences were examined for the presence of variations using Sequencher 4.7 software (Gene Codes Corporation, Ann Arbor, MI, USA), followed by visual inspection. The A of the ATG of the initiator Met codon is denoted nucleotide +1. The nucleotide sequence [NCBI:NM-015277] was used as a reference sequence.(3) The TaqMan SNP Genotyping Assays were performed in 883 subjects aged 30-60 years for genotyping using the method of Taq amplification in the 7900HT Fast Real-Time PCR system (Applied Biosystems). The primers and probes (Applied Biosystems) used in the TaqMan SNP Genotyping Assays were chosen based on information available on the Applied Biosystems Inc. website (http://myscience. appliedbiosystems.com). Finally, all of the three selected representative SNPs were successfully genotyped in 883 subjects participating in the study.(4) Data analyses were performed by SPSS for Windows (version 16; SPSS Inc., Chicago, IL, USA). Values are expressed as the means±SD. The distribution of patient characteristics between the normotensive and hypertensive groups in the Kazakh general population was analyzed using a Student's t-test or a chi-square test. The differences in distributions of genotypes and alleles between the essential hypertension patients and control individuals were analyzed using a chi-square test. In addition, logistic regression analysis was performed to assess the contribution of the major risk factors (including smoking, drinking, age, and obesity). Covariate variance analysis was performed to compare the UNa rate and plasma Na level between the different genotypes after adjusting for age, smoking, and drinking. A case-control-based haplotype study, linkage disequilibrium, and the Hardy-Weinberg equilibrium were analyzed using SNPAlyze, version 7.0 Pro (DYNACOM Co. Ltd., Mobara, Japan). Statistical significance was established at P values< 0.05.Results:(1) Epidemiology research indicates:The age-adjusted prevalence of hypertension in Kazakh was 46.8%. In male, the age-adjusted prevalence of hypertension was higher than female. The prevalence of hypertension was increased with age.(2) Eleven genetic variations in NEDD4L were identified by sequencing 94 hypertensive individuals, among which there were 3 common SNPs with a minor allele frequency> 10%(271420T>C,271454A>G, and 296921-296923delTTG).296921-296923delTTG is a new genetic common variation in NEDD4L which is not found in the NCBI SNP-database. No missense mutations in NEDD4L were identified, but we identified the following two synonymous mutations:R423 (297071G>A in exon 14) with a minor allele frequency of 0.91%; and A944 (351555C>T in exon 30) with a minor allele frequency of 0.56%. None of the variations were in tight linkage disequilibrium with an r2< 0.5. After considering the function and linkage disequilibrium relationships among the identified genetic variations,3 common SNPs with a minor allele frequency of> 10% were selected as representatives for genotyping in Kazakh general population.(3) The distribution of the dominant model (AA vs. AG+GG) of rs2288775, the additive model, and the recessive model (II+ID vs. DD) of 296921-296923delTTG differed significantly between the cases and controls in females (P=0.040, P=0.024, and P=0.007, respectively). After adjusting for confounding factors, logistic regression analysis showed that rs2288775 (in the dominant model) and 296921-296923delTTG (in the recessive model) were significantly associated with hypertension (rs2288775:OR=1.479,95% CI= 1.011-2.064, P=0.044; and 296921-296923delTTG:OR=1.908,95% CI= 1.020-3.568, P =0.043) in females. The frequency of the D-C-G haplotype was significantly higher for cases than for controls in females (P=0.020). For females and total participants, the urinary Na excretion rate was significantly lower in the DD than the I/I+I/D individuals (P=0.032 and P=0.027 respectively). The associations between the SNPs and overweight, dyslipidemia, hyperglycemia, and central obesity were also examined, and no significant associations were found.Conclusions:(1) The age-adjusted prevalence of hypertension was high among Kazakh (aged 30 to 70 years old) from the pasture area of Xinjiang Fukang. Attention should be paid to establishment the ethnic-specific and cost-effective strategy to prevent and treat hypertension efficiently.(2) Eleven genetic variations in NEDD4L were identified, among which there were 3 common SNPs with a minor allele frequency> 10%(271420T>C,271454A>G, and 296921-296923delTTG).296921-296923delTTG is a new genetic common variation in NEDD4L which is not found in the NCBI SNP-database. No missense mutations in NEDD4L were identified. This may be indicative of the relatively high conservation of NEDD4L and the importance of this molecule in regulating salt and water balance and BP. (3) The genetic variations of NEDD4L may be associated with essential hypertension infemales in the Kazakh general population. This reconfirms that genetic variations of NEDD4L might be involved in the pathogenesis of essential hypertension. |
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