Positive Association of the JAG1 rs1327235 Genotype with Coronary Artery Disease in Men,the Tampere Adult Population Cardiovascular Risk Study

Abstract

Aims:

The intronic single nucleotide polymorphism rs1327235 (A>G) close to the JAG gene has been implicated to be involved in blood pressure physiology in a genome-wide association study. We wanted to study whether it was associated with hypertension and coronary artery disease (CAD) in the Tampere adult population cardiovascular risk study.

Materials and Methods:

We analyzed a Finnish periodic health examination cohort of 191 men with diagnosed hypertension and 295 controls. Samples were genotyped for the JAG1 rs1327235 polymorphism using Competitive Allelic Specific PCR (KASP). The incidence of CAD was determined by self-report and the National Hospital Discharge Registry (HILMO).

Results:

There was no association between the JAG1 rs1327235 genotypes with hypertension at the age of 50 years. However, when the subjects were followed to the age of 60 years, those with the genotype GG had a higher prevalence of CAD (17.9%), compared with the A-allele (9.7%) (p = 0.036). When prevalence of CAD was adjusted by body mass index and total cholesterol, the OR for GG genotype was 2.19 (p = 0.029, confidence interval 1.084 - 4.429) compared with A-allele carriers. In addition, the GG genotype was associated with higher total cholesterol and low-density lipoprotein-cholesterol values, compared with the A-allele.

Conclusions:

Our findings suggest that the variations in JAG1 rs1327235 may be involved in CAD and cholesterol metabolism.

Introduction

The Notch signaling pathway is a highly conserved signaling mechanism between ligands on cell membranes and receptors on adjacent cells. JAGGED1 (JAG1) is a ligand for NOTCH transmembrane receptors that is expressed by the JAG1 gene. Binding of JAG1 to the NOTCH receptor results in cleavage of the receptor, and its intracellular domain translocates to the nucleus forming an active transcriptional complex that regulates transcription of target genes. Mutations in JAG1 or NOTCH2 are responsible for the Alagille syndrome, which affects the vasculature and other organs, including liver, heart, and kidney (Gilbert and Spinner, 2017). There are embryonic lethality and vascular defects in mice lacking JAG1 (Xue et al., 1999). Endothelial expression of JAG1 may be required for vascular smooth muscle development (High et al., 2008). A genome-wide association study (GWAS) of systolic and diastolic blood pressure has presented JAG1 as a gene suspected to be involved in blood pressure physiology (International Consortium for Blood Pressure Genome-Wide Association Studies et al., 2011). The blood pressure-associated genetic locus JAG1 rs1327235 was replicated among samples from East African subjects, where it associated with diastolic blood pressure (Kayima et al., 2017). Many of the newly identified variants affecting blood pressure are valid across ethnicities (Ehret and Caulfield, 2013). We wanted to study whether JAG1 rs1327235 associates with hypertension and coronary artery disease (CAD) in the Tampere adult population cardiovascular risk study (TAMRISK).

Materials and Methods

Subjects

TAMRISK study data were collected from periodic health examinations (PHEs) done for 50-year-old men and women living in Tampere, a city in southern Finland with 220,000 inhabitants (Maatta et al., 2015). A public health nurse did the PHE for the study subjects in 2003. Height (cm) and weight (kg) were recorded, and blood pressure measurement (mmHg) was done using a calibrated mercury sphygmomanometer. Serum total cholesterol, high-density lipoprotein-cholesterol, and triglycerides were determined after an overnight fast by standard techniques. The nurse conducted an interview using a structured questionnaire about health and health-related behavior. Current and previous diseases were identified based on self-report, including hypertension, which had been diagnosed by a physician using normal health care procedures. At that time, physicians diagnosed hypertension when blood pressure readings were consistently ≥140/90 mmHg. Separately of the physical examination, we collected during years 2006-2010 from participants by mail buccal swabs for DNA extraction and a permissions form to use PHE information and national registry data. Using the subject's national identity code, data on hospitalizations, including International Classification of Diseases, Tenth Revision codes for discharge diagnoses, were obtained from the National Hospital Discharge Registry (HILMO) maintained by the National Institute of Health and Welfare. Prevalence of ischemic heart diseases (I20-I25) were followed up from 2005 to 2014 until the subjects were on the average 60 years old. In follow-up of the genotyped subjects, there were 54 men with CAD. All participants gave informed consent and the Ethics Committees of the Tampere University Hospital and the City of Tampere approved the study.

Cases (n = 191) were subjects who had hypertension at the age of 50 years (as diagnosed by a physician) and for each case, at least one normotensive control subject (n = 295) was chosen in order of admission from the PHE cohort (n = 6000). The present study population at the age of 50 years thus included 486 men.

Genotyping

DNA was extracted from buccal swabs using a commercial kit (Qiagen, Inc., Valencia, CA). Genotyping was performed using Competitive Allelic Specific Amplification (KASP) genotyping services at KBioscience Institute (United Kingdom).

Statistical analysis

Logistic regression, one-way analysis of variance, or t-test for continuous variables and chi-square test or Fisher's exact test for categorical variables were applied for the comparison of cases, controls, and genotype groups. Analyses were carried out using SPSS 23.0 for Windows (SPSS, Inc., Chicago, IL).

Results

Clinical characteristics of the male group of 191 hypertensive subjects and 295 controls at the age of 50 years are shown in Table 1. Men with hypertension had higher body mass index (BMI), serum triglycerides, systolic- and diastolic blood pressure compared with controls. There was no association of JAG1 rs1327235 genotypes with hypertension. The JAG1 rs1327235 genotype frequencies for the whole study population were AA 31.8%, AG 50.9%, and GG 17.3%. The genotypes were in Hardy-Weinberg equilibrium (χ = 0.159, p > 0.05). Allele frequencies were A 57.2%, and G 42.8%.

Table 1.

Clinical Characteristics of the Male Study Population at the Age of 50 Years Stratified According to Hypertension

	Hypertension (n = 191)	Controls (n = 295)	p ^*
BMI, kg/m² (SD)	29.2 (4.9)	25.9 (3.5)	<0.001
Cholesterol, mmol/L (SD)	5.33 (1.00)	5.32 (0.95)	0.887
HDL-cholesterol, mmol/L (SD)	1.41 (0.36)	1.50 (0.36)	0.091
Triglycerides, mmol/L (SD)	1.75 (1.21)	1.39 (0.90)	0.001
LDL-cholesterol, mmol/L (SD)	3.16 (0.90)	3.21 (0.83)	0.548
Systolic blood pressure, mmHg (SD)	143.8 (16.8)	130.8 (14.7)	<0.001
Diastolic blood pressure, mmHg (SD)	94.0 (9.4)	86.0 (9.0)	<0.001
JAG1 rs1327235 genotypes AA/AG/GG, %	31.4/51.8/16.8	32.0/50.3/17.7	0.942

t-Test or chi-square test.

BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation.

When the subjects were followed up to the age of 60 years, those with genotype GG had higher prevalence of CAD (17.9%), compared with the A-allele (9.7%) (p = 0.036) (Table 2). When prevalence of CAD was adjusted by BMI and total cholesterol determined at the age of 50 years, the OR for GG genotype was 2.19 (p = 0.029, confidence interval 1.084-4.429), compared with A-allele carriers. At the age of 50 years, the GG genotype associated with higher total cholesterol and low-density lipoprotein (LDL)-cholesterol values, compared with the A-allele.

Table 2.

Clinical Characteristics (Means ± Standard Deviation) of the Male Study Population at the Age of 50 Years Stratified According to JAG1 rs1327235 Genotypes

	AA (154)	AG (248)	GG (84)	p-Value^* AA vs. AG vs. GG	p-Value^* AA vs. (AG+GG)	p-Value^* GG vs. (AA+AG)	p-Value^** GG vs. (AA+AG)
CAD, % (n)	11.7 (18)	8.5 (21)	17.9 (15)	0.059	0.759	0.036	0.029
BMI, kg/m² (SD)	27.0 (4.7)	27.4 (4.5)	26.9 (3.8)	0.605	0.515	0.599
Total cholesterol, mmol/L (SD)	5.31 (0.91)	5.24 (0.91)	5.60 (1.18)	0.015	0.855	0.005
HDL-cholesterol, mmol/L (SD)	1.48 (0.36)	1.45 (0.36)	1.50 (0.38)	0.420	0.587	0.331
Triglycerides, mmol/L (SD)	1.52 (1.09)	1.53 (1.04)	1.55 (1.04)	0.985	0.884	0.893
LDL-cholesterol, mmol/L (SD)	3.17 (0.82)	3.13 (0.82)	3.42 (1.01)	0.036	0.693	0.011
Systolic blood pressure, mmHg (SD)	134.9 (18.1)	136.0 (15.7)	137.8 (17.2)	0.453	0.361	0.270
Diastolic blood pressure, mmHg (SD)	88.2 (10.6)	89.5 (9.4)	89.7 (10.1)	0.337	0.142	0.558

p-Values <0.05 are in bold.

Chi-square test, Fisher's exact test, one-way analysis of variance or t-test.

Logistic regression adjusted by BMI and total cholesterol.

CAD, coronary artery disease.

Discussion

A GWAS of systolic and diastolic blood pressure has presented JAG1 as a gene suspected to be involved in blood pressure physiology (International Consortium for Blood Pressure Genome-Wide Association Studies et al., 2011), since the rs1327235 single nucleotide polymorphism (A>G) is located in an intron region, where the nearest gene is JAG1 (Kayima et al., 2017). In fact, results from GWAS have identified a vast number of potentially functional intronic variants affecting disease (Cooper, 2010). However, it may also be possible that rs1327235 is in linkage disequilibrium with a functional polymorphism. In contrast to earlier studies (International Consortium for Blood Pressure Genome-Wide Association Studies et al., 2011; Kayima et al., 2017), we did not find an association of rs1327235 with blood pressure. However, a limitation of our study is that it was restricted to a Finnish population, and the results may not necessarily be extrapolated to other ethnic populations.

The Alagille syndrome is caused by rare and abnormal mutations in JAG1 or the receptor, NOTCH2 (Gilbert and Spinner, 2017). It affects the vasculature among other organs (Gilbert and Spinner, 2017). Earlier studies have found abnormalities of the aorta, pulmonary, intracranial, renal, celiac, superior mesenteric, and subclavian arteries (Kamath et al., 2004). When the men in our study were followed up to the age of 60 years, those with minor genotype GG had higher prevalence of CAD compared with the A-allele. This difference remained even when prevalence of CAD was adjusted by BMI and total cholesterol, suggesting that rs1327235 might be an independent risk factor for this disease.

In addition to systems manifestations of the Alagille syndrome, most of the cases with JAG1 mutations are associated with hypercholesterolemia due to liver function abnormalities (Hannoush et al., 2017; Liu et al., 2018). In line with this finding, the JAG1 rs1327235 minor genotype GG associated with higher serum total cholesterol and LDL-cholesterol values, compared with A-allele carriers, in our male study population.

The JAG1 polymorphism rs1327235 is a DNA sequence variation that is common in the population. When other JAG1 polymorphisms were analyzed in a Mexican population, there was no statistical allele frequency difference identified between patients with Alagille syndrome and controls in 12 previously known polymorphisms, only with one exception, rs2273060 (Vazquez-Martinez et al., 2013). Alagille syndrome with loss of function mutations is at the other end of the spectrum, but it may be that common variations in JAG1 have also potential to compromise health.

Footnotes

Acknowledgments

The expert technical assistance by Mirka Pietiläinen and Nina Peltonen is gratefully acknowledged.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by grants from Competitive research funding of the Pirkanmaa Hospital District.

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