Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with progressive atherosclerosis and incident cardiovascular disease

Abstract

Objective

The enzyme heme oxygenase-1 (HO-1) exerts cytoprotective effects in response to various cellular stressors. A variable number tandem repeat (VNTR) polymorphism in the HO-1 gene promoter region has previously been linked to cardiovascular disease (CVD). We examined this association prospectively in the general population.

Approach and Results

Incidence of stroke, myocardial infarction, or vascular death was registered between 1995 and 2010 in 812 participants of the Bruneck Study aged 45 to 84 years (49.4% males). Carotid atherosclerosis progression was quantified by high-resolution ultrasound. HO-1 VNTR length was determined by polymerase chain reaction. Subjects with ≥32 tandem repeats on both HO-1 alleles compared to the rest of the population (recessive trait) featured substantially increased CVD risk (hazard ratio [95% confidence interval], 5.45 (2.39, 12.42); P<0.0001), enhanced atherosclerosis progression (median difference in atherosclerosis score [interquartile range], 2.1 [0.8, 5.6] vs. 0.0 [0.0, 2.2] mm; P=0.0012), and a trend towards higher levels of oxidised phospholipids on apoB-100 (median OxPL/apoB level [interquartile range], 11364 [4160, 18330] vs. 4844 [3174, 12284] relative light units; P=0.0554). Increased CVD risk in those homozygous for ≥32 repeats was also detected in a pooled analysis of 7848 participants of the Bruneck, SAPHIR, and KORA prospective studies (HR [95% CI], 3.26 [1.50, 7.33]; P=0.0043).

Conclusions

This study found a strong association between the HO-1 VNTR polymorphism and CVD risk confined to subjects with a high number of repeats on both HO-1 alleles, and provides evidence for accelerated atherogenesis and decreased anti-oxidant defence in this vascular high-risk group.

Introduction

Low-grade inflammation, oxidation, and vascular remodelling are cardinal components in the pathophysiology of atherosclerosis¹. Heme oxygenase-1 (HO-1) is the inducible, rate-limiting enzyme of heme degradation and exerts potent anti-inflammatory, anti-oxidative, and anti-apoptotic effects in response to various stressors^2,3. Compelling evidence for a protective effect of HO-1 on the vasculature derives from animal studies demonstrating that HO-1 suppresses the development of atherosclerotic lesions^4–6 and thrombi⁷.Moreover, prominent endothelial damage was observed in rare human HO-1 deficiency⁸ as well as in HO-1 knockout mice⁹.

There is a (GT)_n dinucleotide repeat polymorphism (variable number tandem repeat, VNTR) in the HO-1 gene promoter region, and higher repeat numbers translate into lower enzyme expression^10–13. A deficiency in HO-1-mediated vascular protection in subjects with greater repeat lengths was proposed to predispose to atherosclerosis and its clinical sequelae myocardial infarction (MI) and stroke¹⁴. Studies examining the association between (GT)_n repeat length and cardiovascular disease (CVD) have so far been restricted to selected patient series, mainly subjects admitted for coronary angiography (CAG) or patients with coronary artery disease (CAD) or peripheral arterial disease (PAD), and yielded inconsistent results. A summary of the literature is presented in Table 1. Apart from differences in study design, patient characteristics, and endpoint definitions, heterogeneous results may arise from the different cut-offs applied to categorize repeat number.

Table 1.

Summary of the literature on HO-1 VNTR polymorphism and cardiovascular disease endpoints in humans.

Reference	Primary endpoint	n (cases)	Years of FU	Sample composition	VNTR Cut-off(s) (≥)	Result‡	Effect (short allele)§	Effect (long allele)§
Exner 200123	Restenosis after femoropopliteal BA	96 (23)	0.5	Caucasian, PAD	25 and 29	p	(D) OR 0.2 (0.06, 0.70)
Chen 200211	CAD	796 (474)	CC	Asian, CAG	23 and 32	p		(D) OR 4.7 (1.9, 12.0) in diabetics
Kaneda 200219	CAD	577 (298)	CS	Asian, CAG	27	p	(E) S/S vs L/L: OR 0.23 (0.07, 0.72) in subjects with high cholesterol; OR 0.23 (0.08, 0.71) in diabetics; OR 0.40 (0.17, 0.95) in smokers
Schillinger 200224	AAA, CAD, PAD	271 (210)	CC	Caucasian, vascular risk patients	25	p		(R) more L/L genotype in AAA, p=0.04 NS for CAD, PAD
Chen 200325	Restenosis after coronary stenting, ACE	323 (111)	0.5	Asian, CAD	26	p		(D) OR 3.74 (1.61, 8.70) for stenting (D) OR 3.26 (1.58, 6.72) for ACE
Endler 200426	CAD, MI	649 (438)*	CC	Caucasian, vascular risk patients	25	n	(D) P=0.94
Funk 200427	Ischemic stroke or TIA	797 (399)	CC	Caucasian, stroke	25	p	(E) S/S vs L/L: OR 0.2 (0.1,0.6)
Schillinger 200428	Restenosis after femoropopliteal BA	381 (95)	0.5	Caucasian, PAD	25	p		(R) RR 2.33 (1.41, 4.17), NS for stenting
Dick 200529	MI or PCI or CABG	472 (133)	1.75 (M)	Caucasian, PAD	25	p		(R) HR 2.17 (1.15, 4.17), NS for MACE, all-cause mortality, cerebrovascular events
Gulesserian 200530	Restenosis after coronary stenting	199 (102)	0.5-0.75	Caucasian, CAD	30	p		(D) OR 1.9 (1.0, 3.4), stronger effect in smokers
Li 200531	Restenosis after coronary stenting	187 (52)	0.5	Asian, CAD	30 and 38	n	(D) 30.8% restenosis in S carriers, 22.4% in others; P=0.22
Wijpkema 200615	Restenosis after coronary angioplasty	3146 (287)	0.8 (M)	Caucasian, CAD	25	n	S/L vs. S/S: HR 1.14 (0.90, 1.45); L/L vs. S/S: HR 0.87 (0.55, 1.38)
Tiroch 200717	Restenosis after coronary stenting	1357 (401)	0.5	Caucasian, CAD	25	n	restenosis in 29.2% (S/S), 29.5% (S/L), 29.6% (L/L); P=0.99
Chen 200822	CAD	986 (664)	CS	Asian, CAG	27	p		(R) OR 2.81 (1.22, 6.47) in diabetics; NS with adjustment for ferritin and bilirubin
Lublinghoff 200916	CAD	3219 (2526)†	7.8 (M)	Caucasian, CAG	26 or 28	n	S/L vs. S/S: OR 0.70 (0.49, 1.01); L/L vs. S/S: OR 0.71 (0.49, 1.02)
Bai 201020	Ischemic stroke	347 (183)	CC	Asian, stroke patients and hospital controls	27	p		(M) OR 2.07 (1.07-4.01) in subjects with low HDL
Wu 201032	CVD mortality	504 (22)	10.7 (M)	Asian, arsenic exposure	27	p		(R) OR 2.63 (1.11, 6.25)
Chen 201213	CAD	4596 (2298)	CC	Asian, general population	26	p	(E) S/S vs L/L: OR 0.60 (0.44, 0.81) in subjects with high oxidative stress
Chen 201333	CVD	1080 (307)	4.2 (M)	Asian, hemodialysis	27	p		(R) HR 1.62 (1.28, 2.04)
Gregorek 201334	AAA	234 (117)	CC	Caucasian, AAA patients and hospital controls	25	n	S/L vs. L/L: OR 1.53 (0.90, 3.09); S/S vs. L/L: OR 1.24 (0.87, 1.96)

BA, balloon angioplasty CAD, coronary artery disease; AAA, abdominal aortic aneurysm; ACE, adverse coronary events; MI, myocardial infarction; TIA, transient ischemic attack; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; CVD, cardiovascular disease; FU, follow-up; CC, case-control study; CS, cross-sectional study; (M), median follow-up in years; PAD, peripheral arterial disease; CAG, coronary angiography; OR, odds ratio; RR, risk ratio; HR, hazard ratio; NS, not statistically significant; MACE, major adverse cardiovascular events; HDL, high-density lipoprotein cholesterol.

^*258 MCI and 180 stable CAD

^†2526 CAD and 1339 MI; 752 death

^‡p, positive study – found significant association of HO-1 VNTR length with primary endpoint; n, negative study – did not find significant association of HO-1 VNTR length with primary endpoint.

^§(D), dominant effect, i.e. applies to allele carriers (e.g. pooled S/S and S/L vs. L/L); (E), extreme group comparison (e.g. S/S vs. L/L); (R), recessive effect, i.e. applies to those homozygous for the respective allele (e.g. S/S vs. pooled S/L and L/L);(M), one study applied the cut-off to average within-subject allele length, forming L and S genotypes

We present here the first prospective study on the potential relationship of the HO-1 (GT)_n polymorphism with CVD conducted in the general community.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

HO-1 genotyping resulted in unambiguous results for 812 of 816 subjects for which DNA samples were available (call rate, 99.5%). Duplicate measurement of 95 random DNA samples yielded 100% concordant findings. The distribution of (GT)_n repeat lengths ranged from 12 to 44 repeats and was trimodal, with peaks at 23, 30, and 37 repeats, constituting 20.5%, 40.9%, and 3.9% of alleles (Figure 1). The most common allele combinations were 30/30 and 23/30, observed in n=145 (17.9%) individuals each.

Figure 1.

Joint distribution of heme oxygenase-1 variable number tandem repeat length on each allele. Numbers give the count of subjects that had the corresponding combination of allele lengths. Black lines show the cut-offs we applied to form genotype groups.

Categorization of study subjects by VNTR length (S: <23, M: 23-31, L: ≥32) resulted in only two subjects homozygous for short alleles (SS genotype) and we therefore merged SS and SM genotype groups to form SS/SM (n=35), MM (N=665), ML (n=101), and LL (n=11) groups. Distributions of baseline characteristics according to these four groups are shown in Table 2. Levels of standard risk factors emerged as independent of HO-1 genotype.

Table 2.

Baseline characteristics of the study population according to heme oxygenase-1 genotype.

	SS/SM	MM	ML	LL
n (%)	35 (4.3)	665 (81.9)	101 (12.4)	11 (1.4)
VNTR range (shorter allele)	12 - 22	23 - 31	23 - 31	32 - 37
VNTR range (longer allele)	12 - 31	23 - 31	32 - 44	36 - 38

Baseline characteristics	SS/SM	MM	ML	LL	Pany difference	Ptrend	PLL vs other
Age, years	59.8 ± 11.0	62.9 ± 11.1	62.6 ± 11.1	65.3 ± 9.8	0.368	0.361	0.451
Male sex, n (%)	18 (51.4)	337 (50.7)	41 (40.6)	5 (45.5)	0.293	0.116	0.813
Body mass index, kg/m2	25.2 (23.4, 27.7)	25.3 (23.1, 27.8)	25.7 (23.3, 27.8)	24.5 (22.8, 26.1)	0.694	0.996	0.350
Current smoking, n (%)	8 (22.9)	131 (20.2)	16 (16.0)	1 (9.1)	0.743	0.346	0.458
Diabetes, n (%)	2 (5.7)	74 (11.1)	10 (9.9)	1 (9.1)	0.850	0.970	0.748
Systolic BP, mmHg	147.9 ± 21.3	147.9 ± 20.7	150.9 ± 21.3	147.1 ± 15.4	0.650	0.676	0.650
Diastolic BP, mmHg	87.2 ± 10.1	86.9 ± 9.1	88.2 ± 9.7	87.0 ± 5.3	0.733	0.508	0.927
Total cholesterol, mg/dL	221.7 ± 39.6	229.6 ± 42.9	235.5 ± 42.4	231.6 ± 33.4	0.512	0.187	0.948
HDL cholesterol, mg/dL	59.9 ± 17.5	58.8 ± 16.1	58.1 ± 16.4	56.4 ± 15.4	0.734	0.267	0.567
Ferritin, ng/mL	65 (32, 169)	88 (36, 170)	64 (28, 126)	46 (25, 161)	0.204*	0.194*	0.399*
hsCRP, mg/L	1.9 (0.9, 3.4)	1.6 (0.8, 3.2)	2.0 (1.1, 3.4)	1.8 (1.4, 2.3)	0.098*	0.429*	0.679*

Values are given as n (%), mean ± standard deviation, or median (interquartile range); P_trend is for linear trend; P values are adjusted for age and sex, except those for age and sex, which are only adjusted for the other; VNTR, variable number tandem repeat; S, <23 tandem repeats; M, 23-31 tandem repeats; L, ≥32 tandem repeats

^*variables were log-transformed for significance testing

Crude incidence rates [95% CIs] for CVD were 6.5 [0.0, 15.3], 13.2 [10.8, 15.8], 13.0 [7.1, 19.8], and 65.1 [24.1, 130.4] events per 1000 person-years, for SS/SM, MM, ML, and LL groups, respectively. Accordingly, 55% of subjects in the LL group developed hard CVD endpoints (stroke, MI, or vascular death) in the 15-year follow-up period. Endpoint-specific event counts during the survey period in LL subjects and in other subjects were 4 and 61 for stroke, 2 and 51 for MI, and 0 and 20 for vascular death not due to stroke or MI.

Under adjustment for age and sex, subjects homozygous for the longest repeat lengths (LL) faced a substantially elevated risk for CVD compared to MM subjects (hazard ratio (HR) [95% CI], 5.46 [2.39, 12.50]; P<0.0001) (Table 3). A recessive model best fitted the data and revealed a HR [95% CI] of 5.45 [2.39, 12.42] (P<0.0001) in a comparison of LL to the rest of the study population. Effects remained virtually unchanged under further multivariable adjustment, were similar when excluding 50 subjects with prior CVD (HR [95% CI], 4.44 [1.63, 12.10]; P=0.0036), and were highly significant for the extended CVD endpoint as well (P<0.0001). Analyses of individual disease endpoints yielded a HR [95% CI] of 7.87 [2.84, 21.86] (P<0.0001) for stroke and 2.18 [0.52, 8.96] (P=0.282) for MI.

Table 3.

Associations of heme oxygenase-1 genotype with the primary and extended cardiovascular endpoints.

Primary cardiovascular endpoint
Adjustment →	None		Age and sex		Multivariable*
Repeat length group	HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
SS/SM	0.49 (0.16, 1.55)	0.226	0.62 (0.20, 1.97)	0.420	0.68 (0.21, 2.15)	0.507
MM	1.00 (ref)		1.00 (ref)		1.00 (ref)
ML	0.99 (0.59, 1.67)	0.971	1.15 (0.68, 1.95)	0.599	1.11 (0.65, 1.88)	0.705
LL	4.78 (2.10, 10.88)	<0.001	5.46 (2.39, 12.50)	<0.0001	6.33 (2.74, 14.64)	<0.0001
LL vs. other	4.90 (2.16, 11.13)	<0.001	5.45 (2.39, 12.42)	<0.0001	6.33 (2.75, 14.59)	<0.0001

Extended cardiovascular endpoint
Adjustment →	None		Age and sex		Multivariable*
Repeat length group	HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
SS/SM	0.39 (0.12, 1.23)	0.109	0.47 (0.15, 1.49)	0.202	0.50 (0.16, 1.57)	0.235
MM	1.00 (ref)		1.00 (ref)		1.00 (ref)
ML	1.02 (0.64, 1.64)	0.925	1.20 (0.75, 1.92)	0.455	1.16 (0.72, 1.87)	0.532
LL	5.07 (2.36, 10.88)	<0.0001	5.88 (2.72, 12.68)	<0.0001	6.55 (3.01, 14.30)	<0.0001
LL vs. other	5.21 (2.43, 11.14)	<0.0001	5.87 (2.73, 12.63)	<0.0001	6.56 (3.02, 14.26)	<0.0001

The primary cardiovascular endpoint included non-fatal stroke, non-fatal myocardial infarction, and vascular death. The extended cardiovascular endpoint additionally included peripheral vascular disease and revascularization procedures.

^*Multivariable adjustment was for age, sex, total and high-density lipoprotein cholesterol, current smoking, diabetes mellitus, systolic blood pressure, and body mass index.

HR, hazard ratio.

In sensitivity analyses, we employed penalized cubic splines to examine the precise scale of relationship between VNTR length of each allele and CVD irrespective of pre-defined cut-offs. This gave significant results for the shorter allele (P=0.0073) and provided a post-hoc confirmation of our a priorily fixed cut-off of 32 (Figure 2). When applying alternative and mostly lower cut-offs previously used in the literature (Table 1), findings were not significant, underscoring that high risk was confined to subjects homozygous for the longest HO-1 VNTRs.

Figure 2.

Penalized cubic spline fit of the association of variable number tandem repeat length on the shorter HO-1 allele with the compound CVD endpoint. Grey lines show the cut-offs we applied.

Finally, subjects in the LL group tended to experience atherosclerosis progression (incidence of new plaques or growth of existing ones) more frequently (82% vs 46%, odds ratio [95% CI], 4.72 [0.91, 36.68]; P=0.089) and showed a significantly larger change in the atherosclerosis score over 5 years (median difference in atherosclerosis score [interquartile range], 2.1 [0.8, 5.6] vs. 0.0 [0.0, 2.2] mm; P=0.001), suggesting that the enhanced burden of CVD is at least in part mediated by accelerated atherogenesis. Subjects in the LL group also showed a trend towards elevated baseline levels of OxPL on apoB-100 (median OxPL/apoB levels [interquartile range], 11364 [4160, 18330] vs. 4844 [3174, 12284] relative light units; P=0.055). Results were similar when the Δatherosclerosis score and OxPL/apoB were log-transformed (P=0.014 and P=0.073, respectively). Differences between subjects in the LL group and the rest of the sample with regards to incident CVD, Δatherosclerosis score, and OxPL/apoB are summarized in Figure 3.

Figure 3.

Differences between subjects with the LL genotype and subjects with other genotypes regarding incident cardiovascular disease (1995 to 2010), changes in the carotid atherosclerosis score (1995 to 2000), and OxPL/apoB levels (measured in 1995). Tests were adjusted for age and sex.

We gathered data from three additional prospective cohorts (KORA F3, KORA F4, and SAPHIR) to corroborate our main result. As is visible in Table 4, these cohorts differed in most baseline characteristics. In particular, the additional three cohorts had substantially lower prevalences of the LL genotype, and also substantially lower CVD incidence rates (P=0.011 for heterogeneity after adjustment for age and sex). As a consequence, we were unable to perform a strict independent replication of our key result. However, when pooling data from all four studies, the subjects in the LL group vs. other subjects remained at strongly and significantly elevated risk for CVD (HR [95% CI], 3.26 [1.50, 7.33]; P=0.004; 326 events in 7848 subjects). Moreover, when pooling data from the Bruneck and the SAPHIR study, for which data on an extended endpoint additionally including revascularization procedures and peripheral vascular disease were available, the LL group was also strongly associated with this endpoint (HR [95% CI], 3.98 [1.76, 9.03]; P<0.001; 275 events in 2524 subjects). Both of these associations remained similar and significant under extended multivariable adjustment.

Table 4.

Comparison of prospective cohorts

Study	Bruneck	KORA F3	KORA F4	SAPHIR	Pany difference
N	812	2584	2740	1712
Demographic variables
Age, years	62.73 ± 11.10	56.16 ± 12.53	55.15 ± 12.99	51.38 ± 6.00	<0.0001
Female sex, n (%)	411 (50.6)	1348 (52.2)	1451 (53.0)	635 (37.1)	<0.0001
Metabolic and lifestyle variables
Diabetes, n (%)	87 (10.7)	170 (6.6)	163 (5.9)	54 (3.2)	<0.0001
HDL cholesterol, mg/dL	58.71 ± 16.15	59.08 ± 17.05	56.18 ± 14.42	59.69 ± 15.69	<0.0001
Total cholesterol, mg/dL	230.00 ± 42.56	219.38 ± 39.59	216.28 ± 39.09	228.80 ± 39.97	<0.0001
Systolic blood pressure, mmHg	148.27 ± 20.74	130.16 ± 19.84	121.82 ± 18.32	138.84 ± 17.86	<0.0001
Current smoking, n (%)	156 (19.6)	481 (18.7)	489 (17.8)	332 (19.4)	0.511
Body mass index, kg/m2	25.64 ± 3.84	27.54 ± 4.55	27.44 ± 4.74	26.79 ± 4.12	<0.0001
HO-1 genotype frequencies
S/SML	35 (4.3)	83 (3.2)	65 (2.4)	39 (2.3)	0.001
MM	665 (81.9)	2195 (84.9)	2345 (85.6)	1459 (85.2)
ML	101 (12.4)	298 (11.5)	316 (11.5)	207 (12.1)
LL	11 (1.4)	8 (0.3)	14 (0.5)	7 (0.4)
Incident CVD events, n (%)	132 (16.3)	90 (3.5)	34 (1.2)	70 (4.1)	<0.0001

Values are given as n (%) or as mean ± standard deviation.

HDL, high-density lipoprotein; CVD, cardiovascular disease.

The S/SML genotype group subsumed subjects whose shorter allele had less than 23 tandem repeats.

Discussion

In a prospective cohort study, we observed a substantially increased risk of CVD (hazard ratio [95% confidence interval], 5.45 (2.39, 12.42); P<0.0001) in subjects homozygous for long HO-1 VNTRs, indicating a recessive gene effect. This recessive nature of association is in line with experimental data suggesting the shorter allele to be decisive for HO-1 up-regulation in human umbilical vein endothelial cells (HUVECs)¹⁰. Excess risk in our study was restricted to a small segment of the population (LL genotype, 1.4%).

This is the first prospective study on the relationship of the HO-1 VNTR with CVD conducted in the general population. To the best of our knowledge, the previous studies were conducted in high-risk populations such as patients with pre-existing CVD, coronary stenting, or haemodialysis (Table 1). One Chinese study was population-based but cross-sectional in design¹³. Many of the previous reports on this matter employed lower VNTR cut-offs, most commonly 25 to 27. Of these, three large studies^15–17, including 1800 to 3000 patients, found no relationship between HO-1 VNTR repeat length and their primary endpoints restenosis^15,17 or coronary artery disease¹⁶, but a large number of smaller studies did.Putting these data in perspective with our study, it should be considered that HO-1 induction occurs in response to stress conditions^10,11,18, and a more severe deficit in HO-1 might be necessary in the general (low-risk) population to evoke deleterious effects, whereas a less severe deficit could suffice in higher-risk patients. This interpretation is consistent with several reports that found an association between HO-1 VNTR length and vascular endpoints only in high-risk sub groups such as diabetic subjects or smokers^11,13,19,20.

The dependency of HO-1 protein expression on HO-1 VNTR length has so far been investigated primarily in cell lines. It was found that baseline as well as oxidative stress-induced HO-1 protein levels decreased approximately monotonically parallel to increasing length of the shorter HO-1 allele¹⁰. This extends earlier findings of reduced HO-1 transcriptional activity with increasing VNTR length^11,12. One study found lower increase of HO-1 protein in response to oxidative stress but higher HO-1 baseline expression in cells with long alleles²¹, while another found higher HO-1 protein expression associated with short alleles only under conditions of oxidative stress¹³. There is to date no direct study of this dependency in humans. However, it has been reported that diabetic subjects homozygous for long alleles had increased CAD risk, reduced bilirubin levels, and increased serum ferritin levels, and that the association with CAD risk disappeared with multivariable adjustment for bilirubin and ferritin²². These findings are consistent with reduced HO-1 activity in subjects with long alleles and also with reduced HO-1 activity potentially mediating the effect on CAD risk.

Several lines of evidence suggest that the key finding of our study is valid: (1) The association between HO-1 VNTR and CVD was of particular strength (HR 5.45, lower confidence bound 2.39) and highly significant (P=5.51×10⁻⁵). It would even retain significance in an exploratory setting, testing for all previously used VNTR cut-off values and adjusting for these multiple comparisons (Bonferroni corrected P=4.95×10^-4). (2) The elevated CVD risk observed in the LL HO-1 group was robust in a number of sensitivity analyses (Table 3). (3) The LL group was at elevated CVD risk also in a pooled analysis of 7848 subjects. (4) Vascular protection conferred by HO-1^2,3 is impressively demonstrated by the prominent vascular damage observed in human HO-1 deficiency⁸. (5) The deficit in HO-1 up-regulation in response to cell stress with higher HO-1VNTR number rests on solid experimental evidence^10–13.(6) Subjects with the LL HO-1 genotype in our study had higher levels of OxPL/apoB (P=0.055), which is consistent with decreased HO-1 activity. (7) Finally, we observed a high risk of atherosclerosis progression in the LL HO-1 group, providing a pathophysiological explanation for the elevated CVD risk.

Strengths of our study include its prospective design with long-term high-quality follow-up and representativeness for the general population. Among its weaknesses is the limited number of subjects in extreme repeat length groups, a weakness that extends to the additional population-based cohorts that we employed, which precluded subgroup analyses.

In conclusion, subjects with at least 32 tandem repeats on both HO-1 alleles represent a hitherto neglected vascular high-risk group featured by a substantial burden of CVD, amplified progression of atherosclerosis, and impaired anti-oxidant defence.

Significance.

Heme oxygenase-1 is a key antioxidant and cytoprotective enzyme, and a repeat length polymorphism in its gene promoter region impacts its expression. We found that this polymorphism is associated with cardiovascular risk such that subjects with high repeat lengths on both heme oxygenase-1 alleles suffer a substantially elevated risk. Moreover, we found evidence that oxidative stress and atherosclerosis at least partly mediate this risk elevation. The prospective population-based framework of the Bruneck Study with its high-quality data assessment allowed, for the first time, an investigation of this association both longitudinally and in the general population. This work may delimit a previously underappreciated cardiovascular high-risk group that merits particular preventive attention.

Abbreviations

HO-1

heme oxygenase-1

GT

guanidine thymidine

VNTR

variable number tandem repeat

CVD

cardiovascular disease

OxPL

oxidised phospholipids

ApoB

apolipoprotein B-100