Associations of corin genetic polymorphisms with salt sensitivity, blood pressure changes, and hypertension incidence in Chinese adults

Abstract Corin, a transmembrane serine protease that can cleave pro‐atrial natriuretic peptide (Pro‐ANP) into smaller bioactive molecule atrial natriuretic peptide, has been shown to be involved in the pathophysiology of hypertension, cardiac hypertrophy. We sought to examine the associations of corin genetic variations with salt sensitivity, blood pressure (BP) changes and hypertension incidence. We studied participants of the original Baoji Salt‐Sensitive cohort, recruited from 124 families from seven Chinese villages in 2004 who sequentially received a usual baseline salt diet, a 7‐day low salt diet (3 g/day) and a 7‐day high salt diet (18 g/day), respectively. They were followed up for 8 years (in 2009, 2012) to evaluate the development of hypertension. Corin SNP rs3749584 was significantly associated with diastolic BP (DBP) and mean arterial pressure (MAP) response to low‐salt diet, while rs4695253, rs17654278 were associated with pulse pressure (PP) response to low‐salt diet. SNPs rs4695253, rs12509275, rs2351783, rs2271036, rs2271037 were significantly associated with systolic BP (SBP), DBP, and MAP responses to high‐salt diet. In addition, SNPs rs12641823, rs6834933, rs2271036, and rs22710367 were significantly associated with the longitudinal changes in SBP, DBP, MAP, or PP over 8 years of follow‐up. SNP rs73814824 was significantly associated with the incidence of hypertension over 8 years. Gene‐based analysis showed that corin gene was significantly associated with longitudinal BP changes and hypertension incidence after 8‐year follow‐up. This study suggests that corin may play a role in salt sensitivity, BP progression, and development of hypertension.


INTRODUCTION
Hypertension is determined by environmental factors, genetic factors, and their interactions. 1 Among the environmental determinants of blood pressure (BP) variation, high-dietary salt intake seems to be the most significant. 2 However, BP response to dietary salt intake varies considerably among individuals, a phenomenon known as salt sensitivity. 3 Epidemiological data suggest that individual genetic map may play a crucial role in determining individual BP response to salt intake changes. [4][5][6][7] Therefore, the identification of genetic variants related to salt sensitivity would enhance our understanding of biological mechanisms of BP regulation.
Corin is a transmembrane serine protease of the trypsin superfamily, which can convert pro-atrial natriuretic peptide (Pro-ANP) into atrial natriuretic peptide (ANP) with biological activity. 8 It is mainly expressed in myocardial cells, and also found in kidneys and uterus. 9 In mice, corin deficiency prevents the transformation of Pro-ANP to ANP, leading to hypertension and cardiac hypertrophy. 10 Although the etiology of salt-sensitive hypertension is undoubtedly multifactorial, there is experimental evidence linking corin to the pathogenesis of salt-sensitive hypertension. Wang et al. 11,12 found that corin knockout mice exhibited reduced sodium excretion and impaired natriuretic peptide processing, which maybe an important mechanism in salt-sensitive hypertension. However, the associations of corin with salt sensitivity of BP in humans have not been studied previously.
The human corin gene is composed of 22 exons with a total length of about 200 KB on chromosome 4. 13 Prior studies showed that corin genetic variants were associated with hypertension in African Americans. [14][15][16][17] Mutations that reduce corin activity were also found in families of patients with hypertension and preeclampsia. 18 However, none of previous studies have fully considered the geneenvironment interactions on BP, particularly regarding dietary salt intake. Failure to measure such gene-environment interactions may obscure the genetic contribution to BP variability. Furthermore, no study has yet examined whether genetic variants in the corin gene can predict BP changes or the development of hypertension over time.
In the present study, we aimed to determine the relationships between genetic variations in the corin gene and BP response to strict dietary salt intervention in our previously established cohort. We also used both single marker-based and gene-based analyses to examine the associations of corin gene with longitudinal BP changes and hypertension incidence.

BP measurement and definition of BP response to dietary intervention
BP was measured with a standard mercury sphygmomanometer in the sitting position as previously described. [26][27][28] BP was measured by trained and certified observers during the 3-day baseline observation period and on days 5, 6, and 7of each of the two 7-day intervention periods. Hypertension was defined as systolic BP (SBP) ≥ 140 mmHg, diastolic BP (DBP) ≥ 90 mmHg or use of antihypertensive drugs according to participants' clinical data or self-report. 29 The mean arterial pressure (MAP) was defined as SBP + (2 × DBP). Pulse pressure (PP) was calculated as SBP -DBP.
BP changes from the low-salt intervention to the high-salt intervention may provide a more valid phenotype measure for salt sensitivity as participants' salt intake was controlled during both phases. On the other hand, identifying genetic determinants of BP response to a lowsalt diet from a usual diet should have more direct clinical and public health implications. Therefore, as described previously, we defined BP responses as follows: BP response to high salt = BP on the high-salt diet -BP on the low-salt diet; and BP response to the low-salt = BP on the low-salt diet -BP at baseline. [5][6][7]19

Blood and urinary biochemical analyses
The total cholesterol, triglycerides, high-density lipoprotein (HDL) and fasting glucose levels, urinary concentrations of sodium and potassium were measured by automatic biochemical analyzer (Hitachi, Tokyo, Japan) as previously described. 5

Statistical analyses
The quality control of parental SNP data, including genotyping call rate, Mendelian consistency, MAF and HWE, was performed with PLINK software (version 1.9, http://zzz.bwh.harvard.edu/plink/). We used PLINK software to test the association of single marker with phenotypes, and three genetic models (additive, dominant and recessive) were also assumed for each SNP analysis by mixed-effect regression models. Models were adjusted for the fixed effects of baseline age, sex, and BMI, and the random effect of familial correlations. Bonferroni correction was used for adjustment of multiple testing.
For the analyses of the incidence of hypertension, 51 participants already diagnosed with hypertension at baseline were excluded. We examined the additive association between each SNP and incidence of hypertension using a generalized linear mixed model, which permits multilevel modeling when the response variable follows a binary distribution (eg, incident hypertension). Changes in age, sex, and BMI as fixed effects and familial correlation as a random effect were adjusted in the multivariable analysis by using glmer function in lme4 R package. Gene-based analysis was used to evaluate the overall association of a candidate gene with longitudinal BP changes and hypertension incidence, which was published and internal validity was assessed. 5,7,32,33 Bivariate analysis was performed to examine overall genetic association with longitudinal BP changes and hypertension incidence followed by a multivariate analysis. The truncated product method (TPM), which combines p values from single SNP association analyses, Gene-based analysis was performed using R software (version 3.0.1; http://www. r-project.org).

Baseline characteristics and BP response to dietary intervention
Baseline characteristics and BP responses to low-and high-salt diets in the original cohort of Baoji Salt-Sensitive Study (N = 514) were presented in Table 1. Overall, BP paralleled salt intake, decreased on a lowsalt diet and increased on a high-salt diet. The BP changes during the dietary interventions were greater in probands than in their siblings, offspring, and spouses. Table S1, the urinary sodium excretion significantly decreased from baseline to the low-salt diet, but increased from the low salt to high-salt diet (p < .05), which indicated that the subjects' compliance with the dietary intervention protocol were excellent.

Corin and BP response to dietary intervention
The genome location, MAF, HWE and potential function prediction of each of the SNPs were presented in Supplemental  Table 3 summarizes the characteristics of the subjects at baseline  Table 4.    Table 5. SNP rs73814824 was significantly associated with the hypertension incidence over 8 years. Furthermore, gene-based analysis showed that corin gene was significantly associated with hypertension incidence over 8-year follow-up (p TPM = .0351) after multiple adjustments.

DISCUSSION
In this study, we identified several novel corin SNPs that were significantly associated with longitudinal BP changes and hypertension incidence. The gene-based analyses also showed that croin gene was significantly associated with longitudinal BP changes and the inci- To the best of our knowledge, this is the first study to investigate the associations of corin gene with longitudinal BP changes and hypertension incidence over time. We found that SNPs rs12641823,  11 Wang and associates 12 also showed that BP levels in corin knockout mice on a high-salt diet was significantly increased; however, there was no such change was observed in wild-type mice. It is interesting to speculate how corin affects salt sensitivity by regulating natriuretic peptide system. ANP pathway plays an important role in regulating BP by inhibiting aquaporin 2 (AQP2) and βepithelial Na + channel (β-ENaC). 36 Polzin and associates 37 showed that renal ENaC expression was increased in corin knockout mice, indicating that corin may downregulate renal ENaC expression and activity. Furthermore, Zhao and associates 38 suggested that ANP-mediated inhibition of sodium reabsorption in distal nephron segments was essential to promote natriuresis. In addition, high-salt intake induced an increase in extracellular fluid (ECF). BP may increase because of increased cardiac output and volume-dependent factors. 39,40 As the ECF increased, corin may be activated. Therefore, corin may affect Na + homeostasis or ECF and salt sensitivity of BP through these pathways. Future functional studies are needed to elucidate how the identified risk loci contribute to salt sensitivity of BP at the molecular and cellular level.
The current study has several strengths. First, participation in the dietary interventions was high, and excellent compliance with the study interventions was noted, as evidenced by 24-h urinary sodium excretions during each intervention period. Furthermore, stringent quality control procedures were employed for genotyping and data collection. We used the average of nine separate BP measures at baseline and each follow-up examination, thus reducing measurement errors.
However, this study also has some limitations. Firstly, BP measurement with a mercury sphygmomanometer may cause confounding effect of the white coat phenomena. The study population was relatively small and restricted to northern Chinese individuals. Therefore, the novel findings in our study need to be replicated in other cohorts with different genetic backgrounds. In addition, no washout period was inserted between low-and high salt diets, which may influence the final results.
However, the dietary intervention protocol we adopted was based on The Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study, which was widely used by other researchers. [41][42][43][44] Finally, due to the limited number of genotyped SNPs in corin gene, less frequent genetic variants may have been omitted in the current study.
In conclusion, we report for the first time that corin gene polymorphisms were significantly associated with BP response to dietary salt intervention in Chinese Han population. Using single-marker and genebased analyses, this study further provides direct evidence for the role of corin gene in longitudinal BP phenotypes and hypertension incidence. The findings from the current study provide a basis for potential prevention and a possible therapeutic target for hypertension in the future. In addition, this work contributes to a cumulative understanding of the genomic mechanisms that regulate BP and the development of hypertension.