Association of HLA-DRB1 Single Nucleotide Polymorphisms with Left Ventricular Remodelling in Elderly Patients with Essential Hypertension

Abstract

Objective:

To investigate the associations between human leucocyte antigen DRβ1 (HLA-DRB1) gene polymorphisms and cardiovascular remodelling (left ventricular dilatation [LVD]) in Han Chinese patients ≥ 60 years old, with essential hypertension.

Methods:

LVD was diagnosed via echocardiographic measurements. Multiplex polymerase chain reaction-ligase detection reaction was used to determine the genotypes of three HLA-DRB1 single nucleotide polymorphisms (SNPs; rs2308765, rs9269186 and rs3135388).

Results:

HLA-DRB1 rs2308765 wild-type (G) allele and genotype (GG) were associated with decreased risk of LVD. HLA-DRB1 rs9269186 and rs3135388 were not associated with LVD risk. Mean waist and abdominal circumferences were significantly larger in patients with, compared with those without, LVD. Multivariate logistic regression analyses found that rs2308765 was associated with LVD (GT + TT versus GG; odds ratio 7.958; 95% confidence interval 1.935, 32.723).

Conclusions:

Mutation in HLA-DRB1 rs2308765 was associated with increased risk of LVD in elderly patients with hypertension. Genotype analysis may allow identification of patients at high risk of cardiovascular events such LVD.

Keywords

Cardiovascular remodelling Elderly Essential hypertension Human leucocyte antigen Left ventricular dilatation Single nucleotide polymorphism

Introduction

Left ventricular remodelling occurs during target organ damage that results from essential hypertension,¹ and typically presents as left ventricular hypertrophy and left ventricular dilatation (LVD).² Haemodynamic factors including blood pressure and left ventricular pressure account for < 50% of the between-subject variations in left ventricular mass.^3,4 Nonhaemodynamic factors involved in underlying myocardial hypertrophy include (but are not limited to) duration of hypertension, age, gender and genetic factors.⁵

The human leucocyte antigen (HLA) system includes highly variable coding loci; linkage disequilibrium patterns and allelic frequencies in this region are widely differentiated across broad geographical populations.⁶ Linkage studies have indicated a possible relationship between elements of the HLA system and essential hypertension,^{7 – 9} hypertrophic cardiomyopathy and asymmetric hypertrophy of the myocardium.¹⁰ Certain HLA phenotypes have been found to be associated with LVH and vascular wall hypertrophy in patients with hypertension.^11,12 Associations between HLA gene polymorphisms and cardiovascular remodelling in elderly patients with essential hypertension have not been investigated, however.

The current study investigated the existence of a relationship between three single nucleotide polymorphisms (SNPs) in the HLA DRβ1 (HLA-DRB1) gene (rs2308765, rs9269186 and rs3135388) and cardio -vascular remodelling, in combination with other risk factors such as serum lipid concentrations, in elderly (≥ 60-year-old) Han Chinese patients with essential hypertension.

Patients and methods

Study Population

Patients aged ≥ 60 years, with hypertension (mean systolic blood pressure [SBP] > 140 mmHg and/or diastolic blood pressure [DBP] > 90 mmHg, taken three times on three different days¹³) who attended the Medical Examination Centre of First Hospital, Jilin University (Changchun, China) between May 2009 and August 2009, were recruited to the study. Exclusion criteria were: evidence of secondary forms of hypertension; hyperthyroidism; congenital heart disease; rheumatic heart disease; heart valve disease; physical or mental impairment. Demographic and clinical data were collected via questionnaire and physical examination.

The study protocol was approved by the Institutional Review Board of the First Hospital, Jilin University. All participants provided written informed consent prior to enrolment.

Blood Pressure Measurement

Blood pressure was measured by a skilled health professional using a mercury sphygmomanometer. The mean of two measurements, separated by > 1 min, was recorded as the blood pressure for that day. Patients were enrolled regardless of antihypertensive treatment.

Laboratory Analyses

Blood (4 ml) was collected from patients in the morning after an overnight fast and stored at 4 °C until analysis. Glucose, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, creatinine and triglyceride levels were measured in serum using routine methods at the Clinical Laboratory Department, First Hospital of Jilin University. Creatinine clearance rate (CCR) was estimated using the following formula: CCR (ml/min) = ([140 – age] × weight [kg])/(K × creatinine [mg/dl]), where K = 72 (male) or 85 (female).

Echocardiography

Each patient was examined using two-dimensional targeted M-mode echo -cardiography (iU22 Ultrasound System; Philips Healthcare, Best, Netherlands) by two experienced cardiologists who were unaware of the patient's condition. LVD was diagnosed according to the recommendations of the American Society of Echocardiography.¹⁴ The mean of three measurements was used, and LVD was defined as left ventricular end-diastolic diameter > 55 mm for men and > 50 mm for women.¹⁵ Patients with valvular stenosis or worse than mild valvular regurgitation on colour Doppler echocardiography were excluded from further study.

HLA-DRB1 MPCR–LDR

Genotyping of the HLA-DRB1 locus was performed using multiplex polymerase chain reaction–ligase detection reaction (MPCR–LDR). Whole blood (2 ml) from each patient was collected into sterile tubes containing potassium–ethylene diamine tetra-acetic acid and stored at –80 °C until use. DNA was extracted from the entire sample using the Wizard^® Genomic DNA purification kit (A1125; Promega, Madison, WI, USA). Primers (Table 1) for the three HLA-DRB1 SNP loci (rs2308765, rs1298027 and rs8099917) were designed using Primer Premier 3.0 software (available at: http://frodo.wi.mit.edu/primer3/) and synthesized by Shanghai Technology Services Ltd Synthesis, Shanghai, China. Each 20 μl of reaction mixture contained 50 ng genomic DNA, 2 μl 1 × polymerase chain reaction (PCR) buffer (Promega), 3 mM magnesium, 1 U Taq polymerase (Promega), 4 μl 1 × Q solution (Promega), 2 mM of each dNTP (Promega) and 0.2 pM of primer mix.

Table 1:

Sequences of primers used in polymerase chain reaction analyses for detection of human leucocyte antigen DR β1 (HLA-DRB1) single nucleotide polymorphism (SNP) genotypes, in Han Chinese patients with essential hypertension

SNP	Sense	Antisense	Product length, bp
rs2308765	5′-GGTCACCTCCATGCTGATCT-3′	5′-CAATCCCTTGACCTCGTGAT-3′	157
rs9269186	5′-TGTAGTAATGAATCACAACCAG-3′	5′-GTTTCTCCCTTTCTGAGGGGAA-3′	170
rs3135388	5′-TCCATACCTTGGGGTTTCAG-3′	5′-CATTTGGGCTTGGTCTCATT-3′	164

BP, base pairs.

The cycling programme involved preliminary denaturation at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 30 s, followed by a final elongation step at 72 °C for 7 min. Following amplification, aliquots of each reaction mixture were used for LDR. Each 10 μl LDR mixture contained: 1 μl PCR product (100 ng DNA); 9 μl 1 × LDR buffer (Promega); 12.5 pM probe mixture (Table 2; Shanghai Technology Services Ltd Synthesis); 2 U Taq DNA ligase (New England Biolabs, Beverly, MA, USA); 6.95 μl double-distilled water. The LDR cycling programme involved preliminary denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 s and elongation at 50 °C for 2 min. LDR products were analysed using the ABI 377 Prism^® DNA Sequencer (Applied Biosystems, Foster City, CA, USA).

Table 2:

Sequences of probes used in ligase detection reaction for detection of human leucocyte antigen DRβ 1 (HLA-DRB1) single nucleotide polymorphism (SNP) genotypes in Han Chinese patients with essential hypertension

SNP	Probe sequence	Product length, bp
rs2308765
Common probe	5′-P-TGAAGCCAGAGGATGGAGCAACAAGTTTTTTTTTTTTTTTT-FAM-3′
Wild-type G	5′-TTTTTTTTTTTTTTTTTGCCCATAATCTTACTTGTTCAGC-3′	82
Variant T	5′-TTTTTTTTTTTTTTTTTTTGCCCATAATCTTACTTGTTCAGCA-3′	84
rs9269186
Common probe	5′-P-GGGCATTAAATGGGAATAAAATGGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM-3′
Wild-type C	5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACTGAGAGAAATGAGTTAGTTG-3′	110
Variant G	5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACTGAGAGAAATGAGTTAGTTC-3′	112
rs3135388
Common probe	5′-P-TTGGTTTGTTGGGAGATCTCTACTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM-3′
Wild-type C	5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCATCAGGAAAACCTAAAGTGGGG-3′	118
Variant T	5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCATCAGGAAAACCTAAAGTGGGA-3′	120

BP, base pairs, P, phosphate; FAM, 6-carboxyfluorescein.

Statistical Analyses

Fisher's exact test was used to compare differences in the frequencies of HLA-DRB1 SNP alleles, genotypes and haplotypes between patients with or without LVD. Wilcoxon signed–rank test was used for between-group comparisons of clinical and biochemical characteristics. Clinical and biochemical characteristics (and HLA-DRB1 genotype) were used as independent variables in multivariate logistic regression analyses, to determine the risk factors associated with LVD. A P-value < 0.05 was considered statistically significant. Statistical analyses were performed using SAS^® software, version 8.0 (SAS Institute, Cary, NC, USA).

Results

The study population comprised 148 patients with essential hypertension (140 males/eight females; age range 60 – 97 years; mean age 74 years). The patients were unrelated and originated from the ethnically homogeneous Han population living in Jilin province. Demographic and clinical characteristics of the study population, stratified according to the absence or presence of LVD, are given in Table 3. Mean waist and abdominal circumferences were significantly larger in patients with LVD compared with those without LVD (P = 0.03 and P = 0.02, respectively). There were no other statistically significant between-group differences in demographic or clinical characteristics.

Table 3:

Demographic and clinical characteristics of elderly (≥ 60-year-old) Han Chinese patients with essential hypertension^a recruited to a study investigating the role of human leucocyte antigen DR β1 (HLA-DRB1) single nucleotide polymorphisms in left ventricular remodelling, stratified according to the absence (–) or presence (+)of left ventricular dilatation (LVD)^b

Characteristic	LVD– n = 133	LVD+ n = 15	Statistical significance^c
Gender, males/females	126/7	14/1	NS
Age, years	79.0 (60.0 – 97.0)	81.0 (71.0 – 84.0)	NS
BMI, kg/m²	25.1 (17.1 – 33.1)	26.3 (19.9 – 30.8)	NS
FPG, mmol/l	5.9 (3.6 – 15.0)	6.0 (5.5 – 9.0)	NS
TC, mmol/l	4.4 (3.5 – 6.8)	4.9 (2.8 – 9.2)	NS
Triglyeride, mmol/l	1.3 (0.4 – 9.8)	1.3 (0.6 – 3.5)	NS
HDL, mmol/l	1.3 (0.7 – 2.8)	1.4 (0.9 – 1.8)	NS
LDL, mmol/l	3.1 (1.1 – 6.3)	2.8 (1.8 – 4.3)	NS
Waist circumference, cm	90.2 (11.8 – 118.5)	96.0 (73.0 – 110.0)	P = 0.03
Abdominal circumference, cm	93.0 (11.9 – 117.5)	99.3 (77.0 – 112.0)	P = 0.02
CCR, ml/min	60.9 (17.1 – 119.1)	55.9 (10.6 – 101.0)	NS
Duration of hypertension, years	10.0 (1.0 – 40.0)	16.0 (1.0 – 50.0)	NS
SBP, mmHg	180.0 (148.0 – 240.0)	180.0 (150.0 – 200.0)	NS
DBP, mmHg	100.0 (70.0 – 140.0)	100.0 (90.0 – 120.0)	NS

Data presented as median (range).

Mean systolic blood pressure (SBP) > 140 mm/Hg and/or diastolic blood pressure (DBP) > 90 mm/Hg (measured three times on three different days).¹³

Left ventricular end-diastolic diameter > 55 mm (men) or > 50 mm (women).¹⁵

Wilcoxon signed–rank test.

BMI, body mass index; FPG, fasting plasma glucose; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CCR, estimated creatinine clearance rate.

Genotyping via MPCR–LDR revealed GG or GT genotypes for rs2308765. The rs9269186 genotypes were CC, CG or GG, and the rs3135388 genotypes were CC or CT. Of the three SNPs examined, only rs2308765 was significantly associated with LVD, such that the variant T allele and the GT genotype were associated with an increased risk of LVD (Table 4).

Table 4:

Distribution of human leucocyte antigen DR β1 (HLA-DRB1) single nucleotide polymorphism (SNP) alleles, genotypes and haplotypes in Han Chinese patients with essential hypertension^a, stratified according to the absence (−) or presence (+) of left ventricular dilatation (LVD)^b

SNP	LVD– n = 133	LVD+ n = 15	OR (95% CI)
rs2308765
Allele
G	261 (96.6)	25 (83.3)	1 (reference)
T	9 (3.4)	5 (16.7)	5.71 (1.77, 18.36)
genotype
GG	124 (93.2)	10 (66.7)	1 (reference)
GT or TT	9 (6.8)	5 (33.3)	6.88 (1.93, 24.50)
rs9269186
Allele
C	235 (87)	24 (80.0)	1 (reference)
G	35 (13)	6 (20.0)	1.65 (0.69, 4.32)
genotype
CC	102 (76.7)	10 (66.7)	1 (reference)
CG or GG	31 (23.3)	5 (33.3)	1.64 (0.52, 5.17)
rs3135388
Allele
C	264 (97.8)	29 (96.7)	1 (reference)
T	6 (2.2)	1 (3.3)	1.49 (0.17, 12.84)
genotype
CC	127 (95.5)	14 (93.3)	1 (reference)
CT or TT	6 (4.5)	1 (6.7)	1.51 (0.16, 13.47)
Haplotypes
GCC	220 (82.8)	19 (63.3)	0.28 (0.12, 0.63)
GGC	30 (11.5)	6 (20.0)	1.83 (0.69, 4.84)
TCC	5 (1.8)	4 (13.3)	8.30 (2.05, 33.55)

Data presented as n (%) of alleles or patients.

Mean systolic blood pressure (SBP) > 140 mm/Hg and/or diastolic blood pressure (DBP) > 90 mm/Hg (measured three times on three different days).¹³

Left ventricular end-diastolic diameter > 55 mm (men) or > 50 mm (women).¹⁵

OR, odds ratio; 95% CI, 95% confidence interval.

Analysis of three possible rs2308765/ rs9269186/rs3135388 haplotypes found that patients with the GCC haplotype had a reduced risk of LVD, and that those with the TCC haplotype had an increased risk of LVD (Table 4). The GGC haplotype was not associated with LVD.

Multivariate logistic regression analyses using waist and abdominal circumferences, age, gender and the presence of rs2308765 variant (T) allele as independent variables revealed that only variant rs2308765 was significantly associated with LVD (GT + TT versus GG; odds ratio 7.958; 95% confidence intervals 1.935, 32.723).

Discussion

The current study revealed that waist and abdominal circumference, and HLA-DRB1 SNPs, were associated with left ventricular remodelling in essential hypertension. Epidemiological studies have suggested that age is an important risk factor in myocardial remodelling.^16,17 Age was not significantly associated with LVD in the current study, but this may be explained by the small sample size. Left ventricular remodelling has also been shown to be related to the duration of essential hypertension, gender and hypercholesterolaemia,^{18 – 20} but the current study revealed no such associations.

Studies have shown that both left ventricular cavity size and wall thickness are increased in obese, compared with nonobese, subjects.^{21 – 25} In addition, abdominal circumference has been shown to be associated with increased left ventricular mass in nonobese subjects.²⁶ In the present study, mean abdominal and waist circumferences were significantly larger in patients with LVD than in those without LVD, suggesting a relationship between obesity and left ventricular remodelling.

Specific HLA phenotypes have been associated with increased risk of cardiovascular hypertrophy, including HLA-DR1,¹⁰ DQ7¹¹ and DR11 (in patients with hypertension).^11,12 Based on these studies, the present study investigated the role of HLA-DRB1 in the development of left ventricular remodelling in patients with hypertension. Studies performed in Chinese populations have reported relationships between HLA phenotypes and LVH,such that HLA-DRB15 was associated with increased risk of LVH whereas HLA-DR9 was protective.^19,20 These studies found no relationship between HLA and LVD, however. In the current study, the wild-type G allele and GG genotype of the HLA-DRB1 rs2308765 SNP were associated with a reduced risk of LVD, and the variant GT genotype and T allele were markers of increased LVD risk. The T>G mutation in rs2308765 results in the substitution of wild-type cysteine for phenylalanine. Phenylalanine acts as a substrate for hepatic protein synthesis and transformation into tyrosine that affects the synthesis of certain hormones and neurotransmitters.²⁷ The relationship between this mutation and the pathogenesis of left ventricular remodelling in essential hypertension remains to be elucidated.

In conclusion, HLA-DRB1 rs2308765 was associated with LVD in elderly Han Chinese patients with essential hypertension, and genotypic analysis may help to identify those at high risk of cardiovascular events such as LVD. Further large-scale studies are required in order to confirm these findings in the wider population.

Footnotes

Acknowledgements

This work was supported by a grant from the National Natural Science Foundation of China (No. 30872174).

Conflicts of interest: The authors had no conflicts of interest to declare in relation to this article.

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