GT Microsatellite Repeats in the Heme Oxygenase-1Gene Promoter Associated with Abdominal AorticAneurysm in Croatian Patients

Andrea Crkvenac Gregorek •

Kristina Crkvenac Gornik • Darija Stupin Polancec •

Sanja Dabelic

Received: 5 April 2012 / Accepted: 16 October 2012 / Published online: 21 February 2013

� Springer Science+Business Media New York 2013

Abstract Abdominal aortic aneurysm (AAA) is a complex genetic disorder

caused by the interplay of genetic and environmental risk factors. The number of

(GT)n repeats in the heme oxygenase-1 (HO-1) gene promoter modulates tran-

scription of this enzyme, which might have anti-inflammatory, antioxidant, anti-

apoptotic, and antiproliferative effect. The distribution of alleles and genotypes in

Croatian individuals genotyped for the (GT)n HO-1 polymorphism was similar to

that in other European populations. Frequency of the short (S) alleles (GT \ 25)

was higher in AAA patients (41.9%) than in non-AAA individuals (28.2%,

p = 0.0026) because there were more SL heterozygotes among the AAA patients.

The SL genotype appeared to increase the risk for AAA, but the increase was not

statistically significant after adjustment for age, sex, smoking, hypertension, and

hyperlipidemia (OR = 1.53, 95% CI 0.90–3.09, p = 0.062). These findings con-

tradict those of the only other study performed so far on the association of (GT)n

HO-1 polymorphism and AAA.

Keywords Abdominal aortic aneurysm � Heme oxygenase-1 �Gene polymorphism � GT microsatellite repeats

A. C. Gregorek

Division of Vascular Surgery, Clinic of Surgery, University Hospital Center Zagreb, Zagreb, Croatia

K. C. Gornik

Division of Genetics, Clinic of Pediatrics University Hospital Center Zagreb, Zagreb, Croatia

D. S. Polancec

Galapagos Research Center Ltd, Prilaz baruna Filipovica, Zagreb, Croatia

S. Dabelic (&)

Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry,

University of Zagreb, Ante Kovacica 1, 10000 Zagreb, Croatia

e-mail: sanjad@pharma.hr

123

Biochem Genet (2013) 51:482–492

DOI 10.1007/s10528-013-9579-8

Introduction

Abdominal aortic aneurysm (AAA) is a relatively common, late-onset disease and a

leading cause of sudden death in men older than 55 years. It is defined as a localized

dilatation of the abdominal aorta exceeding the normal diameter (*2 cm) by more

than 50%, and is characterized by chronic aortic wall inflammation, loss of medial

smooth muscle cells, and connective tissue degradation and remodeling. These

pathophysiological events lead to progressive aortic enlargement and ultimately to

rupture, which has a mortality rate exceeding 80%. The best predictor of rupture is

maximal aneurysm diameter, with surgical repair indicated at greater than 5.5 cm.

Abdominal aortic aneurysms are associated with old age, male gender, cigarette

smoking, hypercholesterolemia, and hypertension (Forsdahl et al. 2009). Popula-

tion-based screening with abdominal ultrasound scan reduces the proportion of

aneurysm-related deaths, but adequate pharmacological therapies to attenuate AAA

progression and prevent rupture are still lacking. Elucidation of the biochemical

mechanisms leading to AAA and identification of genes and variants that might

represent risk factors could therefore greatly enhance chances for launching new

drugs and improving efficiency of population-based screening programs. Over the

last decade, candidate-gene association studies and genome-wide association studies

led to the discovery of several genes and sequence variants that are associated with

AAA (Harrison et al. 2012; Hinterseher et al. 2011; Thompson et al. 2008), but the

results of these investigations were often contradictory and should be verified on

larger samples comprising individuals of varying ethnic origins. Until now, only

one study had examined the association of genetic variants in the heme oxygenase-1

(HO-1) gene with AAA (Schillinger et al. 2002).

Heme oxygenase (HO), an enzyme present in three isoforms in mammals,

catalyzes the degradation of heme into biliverdin, releasing free iron and carbon

monoxide. Biliverdin is then rapidly converted into bilirubin, and free iron is

promptly sequestered into ferritin. The three HO isoforms are encoded by different

genes, and although HO-1 is the ‘‘inducible’’ enzyme, the others (HO-2 and HO-3)

are constitutively expressed. Numerous studies support the hypothesis that HO-1

acts as a protective factor, because anti-inflammatory, antioxidant, antiapoptotic,

and antiproliferative effects are observed upon its induction (reviewed in Kim et al.

2011). HO-1 is normally expressed at low levels in most tissues and organs except

for the spleen; it is highly inducible, however, in response to a variety of stimuli,

especially those that increase oxidative stress, and is controlled mostly at the

transcriptional level (Exner et al. 2004b). Within the promoter region of the human

HO-1 gene, mapped to human chromosome 22q12 (Kutty et al. 1994), are binding

sites for numerous transcription factors, such as NF-jB, AP-1, and AP-2 (Exner

et al. 2004b). Several polymorphisms have been detected in the HO-1 gene

promoter region. Three of them have caught the majority of scientific attention: the

(GT)n microsatellite repeat polymorphism and the two single nucleotide polymor-

phisms (SNPs) T(-413)A and G(-1135)A.

Alleles comprising 11–42 GT repeats have been previously detected (Taha et al.

2010) and are usually classified as short (S) or long (L), though standard cutoffs for

grouping the (GT)n repeats have not been determined. In vitro and ex vivo studies

Biochem Genet (2013) 51:482–492 483

123

demonstrated that the number of (GT)n repeats modulates gene transcription: long

(GT)n repeats have been associated with low levels of HO-1 expression in response

to a given stimulus, but the exact mechanism underlying this effect is not known

(Brydun et al. 2007; Hirai et al. 2003; Yamada et al. 2000). Although large-scale

analysis did not confirm a meaningful effect of HO-1 promoter polymorphism on

coronary artery disease or myocardial infarction (Kim et al. 2011), clinical data

from many studies indicate its influence on cardiovascular complications, at least in

some groups of patients (Chen et al. 2012; Endler et al. 2004; Idriss et al. 2008;

Kaneda et al. 2002; Mustafa et al. 2008; Wu et al. 2011).

To investigate the potential association, proposed by the results of Schillinger

et al. (2002), of GT microsatellite repeat polymorphism in the HO-1 gene promoter

with AAA, we set out to determine the frequency of this polymorphism in Croatian

individuals with confirmed and excluded AAA.

Materials and Methods

Patient Selection and Clinical Evaluation

The total study population comprised 234 Croatian inhabitants divided into two

groups: a group of 117 patients with AAA (AAA?) and a control group of 117

patients in whom AAA and any atherosclerotic disease had been excluded (AAA-).

All subjects were patients of the Clinic for Surgery, University Hospital Zagreb,

Croatia. The survey was completely anonymous and was approved by the Ethics

Committee of University Hospital Zagreb and the Ethics Committee of the Medical

Faculty in Zagreb. Written informed consent based on the Helsinki Declaration was

obtained from all volunteers before enrollment. Anamnestic data (age; gender;

smoking; hypertension; diabetes mellitus; hyperlipidemia; associated peripheral,

coronary, and/or carotid disease; aneurysm size; family history) were collected for

all subjects. An aneurysm was defined as a permanent dilatation of the aorta with a

diameter at least 50% greater than that of the proximal neck. The diagnosis was

established by color Doppler ultrasound and computed tomography (CT). Athero-

sclerotic disease of coronary, carotid, and peripheral (lower extremity) arteries was

confirmed or excluded with ultrasound, ECG, and measurement of the ankle-

brachial pressure index, which had to be above 0.9. Subjects were considered to

have arterial hypertension if they were taking antihypertensive medication or had

blood pressure greater than 140/90 mmHg. Hyperlipidemia was defined with total

cholesterol greater than 4.9 mmol/l, LDL cholesterol greater than 3.0 mmol/l, HDL

less than 1 mmol/l, and triglycerides greater than 1.7 mmol/l. Subjects were

considered to have diabetes if they were taking antidiabetic medication or had blood

glucose levels greater than 7 mmol/l in two measurements. Subjects were classified

as smokers only if a recent history of regular cigarette consumption was present. In

the control group, the existence of AAA was excluded by ultrasound or CT. Medical

history and clinical examination excluded the presence of atherosclerotic disease of

coronary, carotid, and peripheral arteries.

484 Biochem Genet (2013) 51:482–492

123

DNA Extraction and Genotyping

Peripheral blood samples were collected by venipuncture in EDTA-containing tubes.

DNA was isolated from whole blood with a NucleoSpin Blood Kit (Macherey–Nagel,

Duren, Germany) following the manufacturer’s protocol. PCR amplification of the 50

flanking region containing the poly-GT sequence of the HO-1 gene promoter was

performed as described by Kimpara et al. (1997). The PCR products were analyzed

using a laser-based automated DNA sequencer (ALFexpress, Pharmacia Biotech,

Uppsala, Sweden). The length of each (GT)n repeat allele was calculated using two

alleles as size markers, the repeat numbers of which were 23 and 30, and a

commercially available size standard (Sizer 200, Amersham Pharmacia, Cardiff, UK),

using AlleleLocator analysis software (Amersham Pharmacia). To verify the accuracy

of the (GT)n repeat determination, a commercially available service (Macrogen,

Korea) analyzed the sequences of 10 randomly chosen samples. The sequencing

results confirmed the results of the genotyping.

Statistical Analysis

Continuous data were reported as the median and interquartile range (IQR, 25th–75th

percentile). Discrete data were reported as counts and frequencies. Groups of

continuous data were compared by the t-test or, as appropriate, by the Mann–

Whitney U test. Frequencies between groups and Hardy–Weinberg equilibrium

were subjected to the chi-square test. The association of specific genotypes with

AAA was determined by logistic regression analysis adjusted for potentially

confounding effects of baseline variables. The level of significance was set at 0.05.

Results

Altogether, 234 subjects were included in this study. Relative to the AAA- control

group, the AAA? group contained older subjects; had a significantly greater

proportion of subjects with hypertension, hyperlipidemia, and diabetes mellitus; and

had more males and more smokers (Table 1). Additionally, the AAA? group

contained 59 subjects (50.4% of all AAA patients) with one or more types of

atherosclerotic disease (coronary artery disease, peripheral arterial disease, and

coronary artery stenosis), whereas such subjects were not present in the control

group. The median maximum diameter of the aortic aneurysm was 6.0 cm (IQR

5.0–7.1), and 80 patients (68.4%) had an aneurysm 5.5 cm or greater in diameter.

Six patients (5.1%) had a ruptured aneurysm, and an additional nine patients (7.7%)

had a symptomatic aneurysm, with a tendency for rupture.

Repeat-length analysis showed that the number of (GT)n repeats ranged from 21

to 37 in controls and from 22 to 38 in AAA patients. Alleles with 35 and 36 repeats

were not detected in the studied cohorts. The most common alleles among both

groups were (GT)23 and (GT)30 (Fig. 1). The alleles were classified into short

(S) and long (L) groups, with fewer than 25 repeats in class S alleles and 25 or more

repeats in class L alleles. According to this classification, 35% of the studied

Biochem Genet (2013) 51:482–492 485

123

Croatian individuals were carriers of the S allele, and 65% were carriers of the L

allele, with a genotype distribution of 12.4% SS, 45.3% SL, and 42.3% LL

(Table 2). The chi-square test for independence between allelic classes confirmed

Hardy–Weinberg equilibrium in the overall study group (v2 = 0.088, p = 0.766),

as well as in the AAA? (p = 0.181) and AAA- (0.219) groups.

The difference in allelic distribution between the AAA patients and controls was

statistically significant (p = 0.0026): the S allele was found in 41.9% of AAA patients

and only 28.2% of the control group (OR = 1.86, 95% CI 1.24–2.55, p = 0.007); after

adjustment for age, sex, smoking, hypertension, and hyperlipidemia, however, the

difference was not significant (OR = 1.63, 95% CI 1.05–2.26, p = 0.055). The rarest

genotype, SS, was present in similar frequencies in both AAA patients (14.5%) and

Table 1 Characteristics of study subjects with and without AAA

Variable Total (N = 234) AAA? (N = 117) AAA- (N = 117) p

Age (IQR) 66 (65–68) 69 (62–73) 64 (57–73) 0.0040

Men 169 (72.2%) 102 (87.2%) 67 (57.3%) \ 0.0001

Smoking 67 (28.6%) 53 (45.3%) 14 (12.0%) \ 0.0001

Hypertension 104 (44.4%) 93 (52.0%) 11 (9.4%) \ 0.0001

Diabetes mellitus 14 (6.0%) 14 (12.0%) 0 \ 0.0001

Hyperlipidemia 67 (28.6%) 65 (55.6%) 2 (1.7%) \ 0.0001

Coronary artery disease 43 (18.4%) 43 (36.8%) 0 \ 0.0001

Peripheral arterial disease 18 (7.7%) 18 (15.4%) 0 \ 0.0001

Coronary artery stenosis 18 (7.7%) 18 (15.4%) 0 \ 0.0001

Family history of AAA 12 (5.1%) 12 (10.3%) 0 \ 0.0001

IQR interquartile range, p statistically significant at p \ 0.05

Alle

le fr

eque

ncy

(%)

Number of (GT)n repeats

0

5

10

15

20

25

30

35

40

45

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

total

AAA+

AAA-

Fig. 1 Allele frequency of the (GT)n repeat polymorphism in the heme oxygenase-1 gene promoter inthe study population of Croatian individuals. Black bars, all subjects (N = 234); light gray bars, subjectswith AAA (AAA?; N = 117); dark gray bars, subjects without AAA (AAA-; N = 117)

486 Biochem Genet (2013) 51:482–492

123

controls (10.3%, p = 0.4376) and was not associated with increased risk for AAA

(p [ 0.05). SL heterozygotes were more frequent among AAA patients (54.7%) than

among controls (35.9%, p = 0.0058), and LL homozygotes were more prevalent

among controls (53.8%) than among AAA patients (30.8%, p = 0.0006). The SL

genotype was significantly associated with increased risk for developing AAA

(OR = 1.72, 95% CI 1.20–2.81, p = 0.009). In a logistic regression model, however,

including age, sex, smoking, hypertension, hyperlipidemia, and the HO-1 genotype as

covariates, the SL association with increased risk for AAA was not statistically

significant (OR = 1.53, 95% CI 0.90–3.09, p = 0.062; Table 2). No difference in

allele or genotype frequencies was observed between AAA patients who smoked or

had hypertension, hyperlipidemia, or atherosclerotic disease and AAA patients

without these conditions. Additionally, we found no correlation between AAA patient

genotype and aneurysm size (Table 3).

Discussion

Abdominal aortic aneurysm is a life-threatening disease. Its formation and

progression are determined by both environmental and genetic factors, though the

genetic predisposition for AAA is mostly unknown. An association with AAA has

been shown for several genes and genetic variants in more than a few previous

studies, but until now, only a single study had examined the relationship between

(GT)n repeats in the HO-1 gene promoter and the risk for developing AAA

(Schillinger et al. 2002).

Our study was carried out on individuals of Croatian origin. To the best of our

knowledge, this is the first report on the frequency of (GT)n microsatellite repeats of

Table 2 Distribution of (GT)n HO-1 alleles and genotypes among subjects with and without AAA

Participant group Allele Genotype

S L SS SL LL

Total (N = 234) 164 (35.0%) 304 (65.0%) 29 (12.4%) 106 (45.3%) 99 (42.3%)

AAA? (N = 117) 98 (41.9%) 136 (58.1%) 17 (14.5%) 64 (54.7%) 36 (30.8%)

AAA- (N = 117) 66 (28.2%) 168 (71.8%) 12 (10.3%) 42 (35.9%) 63 (53.8%)

p value 0.0026 0.0026 0.4376 0.0058 0.0006

Crude OR 1.86 1.00 1.44 1.72 1.00

95% CI 1.24–2.55 0.91–2.11 1.20–2.81

p value 0.007 0.189 0.009

Adjusted OR 1.63 1.00 1.24 1.53 1.00

95% CI 1.05–2.26 0.87–1.96 0.90–3.09

p value 0.055 0.125 0.062

p statistically significant at p \ 0.05

S (short) alleles \ 25 GT repeats; L (long) alleles C 25 GT repeats. Adjusted OR, odds ratio adjusted for

age, sex, smoking, hypertension, and hyperlipidemia; allele L and genotype LL were regarded as ref-

erence points

Biochem Genet (2013) 51:482–492 487

123

the HO-1 gene in the Croatian population. Overall, 16 alleles have been detected,

and the distribution of the numbers of (GT)n repeats was bimodal, with one peak at

23 GT repeats and the other peak at 30 GT. Our distribution results are comparable

to those reported for other Caucasian populations (Chen et al. 2002; Denschlag et al.

2004; Dick et al. 2005; Exner et al. 2004a; Katana et al. 2010, 2011; Rueda et al.

2007). No consensus has been achieved on the optimum cutpoint for the (GT)n

repeat-length polymorphism, so it is difficult to compare results among investiga-

tions, especially taking into account that some investigators divide alleles into two

groups (short and long) and others into three groups (short, medium, long).

The main aim of our study was to assess the association of the (GT)n HO-1 gene

promoter polymorphism and AAA, so we chose the same classification that was

applied in the only previous study regarding the relationship of (GT)n repeats and

AAA (Schillinger et al. 2002). In our study, however, the previous finding that

carriers of short HO-1 (GT)n repeats are less frequent among AAA patients was not

replicated. On the contrary, short alleles were found more often among individuals

with AAA. Genetic association studies are difficult to replicate, particularly in

Table 3 Association of aneurysm size and other covariates with (GT)n HO-1 alleles and genotypes

among AAA patients

Covariate Allele Genotype

S L SS SL LL

Aneurysm size

Small (N = 37, 31.6%) 33 (44.6%) 41 (55.4%) 5 (13.5%) 23 (62.2%) 9 (24.3%)

Large (N = 80, 68.4%) 65 (40.6%) 95 (59.4%) 12 (15.0%) 41 (51.3%) 27 (33.8%)

p value 0.6641 0.6641 0.9458 0.3672 0.4124

Atherosclerotic disease

Yes (N = 59, 51.4%) 49 (41.53%) 69 (58.47%) 7 (11.86%) 35 (59.32%) 17 (28.81%)

No (N = 58, 49.6%) 49 (42.24%) 67 (57.76%) 10 (17.24%) 29 (50%) 19 (32.76%)

p value 0.9981 0.9821 0.5732 0.4083 0.7929

Hypertension

Yes (N = 93, 79.5%) 77 (41.4%) 109 (58.6%) 12 (12.9%) 53 (57.0%) 28 (30.0%)

No (N = 24, 20.5%) 21 (43.8%) 27 (56.5%) 5 (20.8%) 11 (45.8%) 8 (33.3%)

p value 0.8915 0.9212 0.5128 0.4516 0.9487

Hyperlipidemia

Yes (N = 65, 55.5%) 53 (40.8%) 77 (59.2%) 10 (15.4%) 33 (50.8%) 22 (33.8%)

No (N = 52, 45.5%) 45 (43.3%) 59 (56.7%) 7 (13.5%) 31 (59.6%) 14 (26.9%)

p value 0.8012 0.8012 0.9794 0.4453 0.5471

Smoking

Yes (N = 53, 45.3%) 48 (45.3%) 58 (54.7%) 10 (18.9%) 28 (52.8%) 15 (28.3%)

No (N = 64, 54.7%) 50 (39.1%) 78 (60.9%) 7 (10.9%) 36 (56.3%) 21 (32.8%)

p value 0.4101 0.4101 0.3376 0.8477 0.7461

p statistically significant at p \ 0.05

Aneurysm size, small \ 5.5-cm diameter; large C 5.5-cm diameter. Atherosclerotic disease includes

coronary artery disease, peripheral arterial disease, and coronary artery stenosis

488 Biochem Genet (2013) 51:482–492

123

multifactorial conditions such as AAA. In interpreting the findings from different

populations of patients with the same disease outcome, one has to be aware of the

intrinsic complexity of genetic association studies. Many human diseases exhibit

complicated clinical phenotypes that are influenced by the interactions of multiple

genes, environmental factors, and treatment. Differences in findings among studies

may arise from several undetectable causes. Nevertheless, one important limitation

of both studies investigating the (GT)n HO-1 polymorphism and AAA is the

relatively small patient cohort: 70 AAA patients in the study of Schillinger et al.

(2002) and 117 AAA patients in our study. Additionally, the 95% confidence

intervals for the odds ratios are relatively wide, so the conclusions stated might be

just accidental findings and thereby give rise to numerous possibilities for error.

Though the upregulated expression of HO-1 (which might be enabled by the

presence of short (GT)n repeats in the HO-1 promoter) is generally considered to

protect against the development of a whole range of diseases, there are some

possible explanations for an association of S alleles with AAA. The mechanisms

underlying the HO-1 induction by its multiple inducers are complex and are tightly

regulated at the transcriptional level. At present, the exact molecular mechanism by

which the (GT)n polymorphism is able to modify HO-1 promoter activity remains

unclear. One hypothesis is that (GT)n repeats cause conformational changes in

DNA, thus negatively affecting transcriptional activity. Another possibility is that

the (GT)n microsatellites are in linkage disequilibrium with SNP T(-413)A, which

may actually exert the functional effect (Ono et al. 2004). Besides modifier genetic

variants, other nongenetic confounders, such as vitamin E levels, are known to

functionally inhibit HO-1 mRNA and influence the expression of HO-1 (Jenkins

et al. 2001).

Paradoxically, HO-1 inducers could be both stimulators and inhibitors of a

particular disease process. For example, proatherogenic stimuli such as TNF-a,

lipopolysaccharide, and hypoxia or antiatherogenic stimuli such as IL-10 have been

reported to induce HO-1 (Kacimi et al. 2000; Lee and Chau 2002; Terry et al. 1998).

Additionally, short repeats have been associated with increased risk for several

conditions, such as various types of tumors (Vashist et al. 2011a, b), hypertension

(Lin et al. 2011), idiopathic recurrent miscarriage (Denschlag et al. 2004), acute

respiratory distress syndrome (Sheu et al. 2009), and childhood-onset systemic

lupus erythematosus (Cordova et al. 2012), and some of these conditions, as well as

AAA, are thought to have inflammation in their background. Moreover, not all

inflammation-related diseases are associated with (GT)n repeat polymorphism

(Hausmann et al. 2008). Although HO-1 generally acts as an anti-inflammatory

molecule, there are some reports of HO-1-mediated pro-inflammatory actions under

certain conditions. Tamion et al. (1999) describe the induction of TNF-a and IL-6

by HO-1 in macrophages exposed to hypoxia and subsequent reoxygenation, and

Kanakiriya et al. (2003) demonstrate that HO-1 elicits the expression of monocyte

chemoattractant protein-1 in renal tubular epithelial cells.

Moreover, the higher frequency of the S allele in AAA patients observed in this

study is due to the much higher frequency of SL heterozygotes; the number of SS

homozygotes was higher among AAA patients than among healthy controls but was

not statistically significant. The elucidation of that confusing finding is another

Biochem Genet (2013) 51:482–492 489

123

challenge for future investigations; whether it lies in epigenetic regulation,

autoregulation of gene expression, selective advantage, or some other factors needs

to be clarified. That result should be interpreted with caution, however, because SS

homozygotes are found in relatively small frequency (*12%) in the general

Croatian population, and an association study on a larger number of individuals

might turn the nonsignificant higher frequency into a statistically significant result.

In this study we found no correlation between aneurysm size and (GT)n genotype.

Additionally, we found no association between (GT)n genotype and the presence of

atherosclerotic diseases, hyperlipidemia, hypertension, or smoking among AAA

patients, suggesting that the existence of the S allele is specifically associated with

AAA and not with these other conditions, yielding falsely positive results. We can

conclude that the role of the HO-1 (GT)n repeat promoter polymorphism in AAA

formation and progression remains unclear and needs further investigation.

Acknowledgments This work was supported by grant 006-006-1194-1218 from the Ministry of

Science, Education and Sports of the Republic of Croatia.

References

Brydun A, Watari Y, Yamamoto Y, Okuhara K, Teragawa H, Kono F, Chayama K, Oshima T, Ozono R

(2007) Reduced expression of heme oxygenase-1 in patients with coronary atherosclerosis.

Hypertens Res 30:341–348

Chen M, Zhou L, Ding H, Huang S, He M, Zhang X, Cheng L, Wang D, Hu FB, Wu T (2012) Short

(GT)n repeats in heme oxygenase-1 gene promoter are associated with lower risk of coronary heart

disease in subjects with high levels of oxidative stress. Cell Stress Chaperones 17:329–338

Chen YH, Lin SJ, Lin MW, Tsai HL, Kuo SS, Chen JW, Charng MJ, Wu TC, Chen LC, Ding YA, Pan

WH, Jou YS, Chau LY (2002) Microsatellite polymorphism in promoter of heme oxygenase-1 gene

is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum Genet

111:1–8

Cordova EJ, Martinez-Hernandez A, Ramirez-Bello J, Velazquez-Cruz R, Centeno F, Baca V, Orozco L

(2012) HMOX1 promoter (GT)n polymorphim is associated with childhood-onset systemic lupus

erythematosus but not with juvenile rheumatoid arthritis in a Mexican population. Clin Exp

Rheumatol 30:297–301

Denschlag D, Marculescu R, Unfried G, Hefler LA, Exner M, Hashemi A, Riener EK, Keck C, Tempfer

CB, Wagner O (2004) The size of a microsatellite polymorphism of the haem oxygenase 1 gene is

associated with idiopathic recurrent miscarriage. Mol Hum Reprod 10:211–214

Dick P, Schillinger M, Minar E, Mlekusch W, Amighi J, Sabeti S, Schlager O, Raith M, Endler G,

Mannhalter C, Wagner O, Exner M (2005) Haem oxygenase-1 genotype and cardiovascular adverse

events in patients with peripheral artery disease. Eur J Clin Investig 35:731–737

Endler G, Exner M, Schillinger M, Marculescu R, Sunder-Plassmann R, Raith M, Jordanova N, Wojta J,

Mannhalter C, Wagner OF, Huber K (2004) A microsatellite polymorphism in the heme oxygenase-

1 gene promoter is associated with increased bilirubin and HDL levels but not with coronary artery

disease. Thromb Haemost 91:155–161

Exner M, Bohmig GA, Schillinger M, Regele H, Watschinger B, Horl WH, Raith M, Mannhalter C,

Wagner OF (2004a) Donor heme oxygenase-1 genotype is associated with renal allograft function.

Transplantation 77:538–542

Exner M, Minar E, Wagner O, Schillinger M (2004b) The role of heme oxygenase-1 promoter

polymorphisms in human disease. Free Radical Biol Med 37:1097–1104

Forsdahl SH, Singh K, Solberg S, Jacobsen BK (2009) Risk factors for abdominal aortic aneurysms: a

7-year prospective study: the Tromso Study, 1994–2001. Circulation 119:2202–2208

Harrison SC, Kalea AZ, Holmes MV, Agu O, Humphries SE (2012) Genomic research to identify novel

pathways in the development of abdominal aortic aneurysm. Cardiol Res Pract 2012:852829.

doi:10.1155/2012/852829

490 Biochem Genet (2013) 51:482–492

123

Hausmann M, Paul G, Kellermeier S, Frey I, Scholmerich J, Falk W, Menzel K, Fried M, Herfarth H,

Rogler G (2008) (GT)n dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is

not associated with inflammatory bowel disease risk or disease course. Clin Exp Immunol

153:81–85

Hinterseher I, Tromp G, Kuivaniemi H (2011) Genes and abdominal aortic aneurysm. Ann Vasc Surg

25:388–412

Hirai H, Kubo H, Yamaya M, Nakayama K, Numasaki M, Kobayashi S, Suzuki S, Shibahara S, Sasaki H

(2003) Microsatellite polymorphism in heme oxygenase-1 gene promoter is associated with

susceptibility to oxidant-induced apoptosis in lymphoblastoid cell lines. Blood 102:1619–1621

Idriss NK, Blann AD, Lip GY (2008) Hemoxygenase-1 in cardiovascular disease. J Am Coll Cardiol

52:971–978

Jenkins JK, Huang H, Ndebele K, Salahudeen AK (2001) Vitamin E inhibits renal mRNA expression of

COX II, HO I, TGFbeta, and osteopontin in the rat model of cyclosporine nephrotoxicity.

Transplantation 71:331–334

Kacimi R, Chentoufi J, Honbo N, Long CS, Karliner JS (2000) Hypoxia differentially regulates stress

proteins in cultured cardiomyocytes: role of the p38 stress-activated kinase signaling cascade, and

relation to cytoprotection. Cardiovasc Res 46:139–150

Kanakiriya SK, Croatt AJ, Haggard JJ, Ingelfinger JR, Tang SS, Alam J, Nath KA (2003) Heme: a novel

inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am J Physiol Renal

Physiol 284:F546–F554

Kaneda H, Ohno M, Taguchi J, Togo M, Hashimoto H, Ogasawara K, Aizawa T, Ishizaka N, Nagai R

(2002) Heme oxygenase-1 gene promoter polymorphism is associated with coronary artery disease

in Japanese patients with coronary risk factors. Arterioscler Thromb Vasc Biol 22:1680–1685

Katana E, Skoura L, Giakoustidis D, Takoudas D, Malisiovas N, Daniilidis M (2010) Association

between the heme oxygenase-1 promoter polymorphism and renal transplantation outcome in

Greece. Transplant Proc 42:2479–2485

Katana EP, Skoura LG, Scouras ZG, Daniilidis MA (2011) Genotype and allele frequencies of heme

oxygenase-1 promoter region in a Greek cohort. Chin Med J (Engl) 124:3408–3411

Kim YM, Pae HO, Park JE, Lee YC, Woo JM, Kim NH, Choi YK, Lee BS, Kim SR, Chung HT (2011)

Heme oxygenase in the regulation of vascular biology: from molecular mechanisms to therapeutic

opportunities. Antioxid Redox Signal 14:137–167

Kimpara T, Takeda A, Watanabe K, Itoyama Y, Ikawa S, Watanabe M, Arai H, Sasaki H, Higuchi S,

Okita N, Takase S, Saito H, Takahashi K, Shibahara S (1997) Microsatellite polymorphism in the

human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer

and Parkinson disease. Hum Genet 100:145–147

Kutty RK, Kutty G, Rodriguez IR, Chader GJ, Wiggert B (1994) Chromosomal localization of the human

heme oxygenase genes: heme oxygenase-1 (HMOX1) maps to chromosome 22q12 and heme

oxygenase-2 (HMOX2) maps to chromosome 16p13.3. Genomics 20:513–516

Lee TS, Chau LY (2002) Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in

mice. Nat Med 8:240–246

Lin R, Fu W, Zhou W, Wang Y, Wang X, Huang W, Jin L (2011) Association of heme oxygenase-1 gene

polymorphisms with essential hypertension and blood pressure in the Chinese Han population.

Genet Test Mol Biomarkers 15:23–28

Mustafa S, Weltermann A, Fritsche R, Marsik C, Wagner O, Kyrle PA, Eichinger S (2008) Genetic

variation in heme oxygenase 1 (HMOX1) and the risk of recurrent venous thromboembolism. J Vasc

Surg 47:566–570

Ono K, Goto Y, Takagi S, Baba S, Tago N, Nonogi H, Iwai N (2004) A promoter variant of the heme

oxygenase-1 gene may reduce the incidence of ischemic heart disease in Japanese. Atherosclerosis

173:315–319

Rueda B, Oliver J, Robledo G, Lopez-Nevot MA, Balsa A, Pascual-Salcedo D, Gonzalez-Gay MA,

Gonzalez-Escribano MF, Martin J (2007) HO-1 promoter polymorphism associated with rheumatoid

arthritis. Arthritis Rheum 56:3953–3958

Schillinger M, Exner M, Mlekusch W, Domanovits H, Huber K, Mannhalter C, Wagner O, Minar E

(2002) Heme oxygenase-1 gene promoter polymorphism is associated with abdominal aortic

aneurysm. Thromb Res 106:131–136

Sheu CC, Zhai R, Wang Z, Gong MN, Tejera P, Chen F, Su L, Thompson BT, Christiani DC (2009)

Heme oxygenase-1 microsatellite polymorphism and haplotypes are associated with the develop-

ment of acute respiratory distress syndrome. Intensive Care Med 35:1343–1351

Biochem Genet (2013) 51:482–492 491

123

Taha H, Skrzypek K, Guevara I, Nigisch A, Mustafa S, Grochot-Przeczek A, Ferdek P, Was H,

Kotlinowski J, Kozakowska M, Balcerczyk A, Muchova L, Vitek L, Weigel G, Dulak J, Jozkowicz

A (2010) Role of heme oxygenase-1 in human endothelial cells: lesson from the promoter allelic

variants. Arterioscler Thromb Vasc Biol 30:1634–1641

Tamion F, Richard V, Lyoumi S, Hiron M, Bonmarchand G, Leroy J, Daveau M, Thuillez C, Lebreton JP

(1999) Induction of haem oxygenase contributes to the synthesis of pro-inflammatory cytokines in

re-oxygenated rat macrophages: role of cGMP. Cytokine 11:326–333

Terry CM, Clikeman JA, Hoidal JR, Callahan KS (1998) Effect of tumor necrosis factor-alpha and

interleukin-1 alpha on heme oxygenase-1 expression in human endothelial cells. Am J Physiol

274:H883–H891

Thompson AR, Drenos F, Hafez H, Humphries SE (2008) Candidate gene association studies in

abdominal aortic aneurysm disease: a review and meta-analysis. Eur J Vasc Endovasc Surg

35:19–30

Vashist YK, Uzungolu G, Kutup A, Gebauer F, Koenig A, Deutsch L, Zehler O, Busch P, Kalinin V,

Izbicki JR, Yekebas EF (2011a) Heme oxygenase-1 germ line GTn promoter polymorphism is an

independent prognosticator of tumor recurrence and survival in pancreatic cancer. J Surg Oncol

104:305–311

Vashist YK, Uzunoglu G, Deutsch L, Kalinin V, Zehler O, Alzadjali A, Kutup A, Izbicki JR, Yekebas EF

(2011b) Heme oxygenase-1 promoter polymorphism is a predictor of disease relapse in pancreatic

neuroendocrine tumors. J Surg Res 166:e121–e127

Wu MM, Chiou HY, Chen CL, Hsu LI, Lien LM, Wang CH, Hsieh YC, Wang YH, Hsueh YM, Lee TC,

Cheng WF, Chen CJ (2011) Association of heme oxygenase-1 GT-repeat polymorphism with blood

pressure phenotypes and its relevance to future cardiovascular mortality risk: an observation based

on arsenic-exposed individuals. Atherosclerosis 219:704–708

Yamada N, Yamaya M, Okinaga S, Nakayama K, Sekizawa K, Shibahara S, Sasaki H (2000)

Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with

susceptibility to emphysema. Am J Hum Genet 66:187–195

492 Biochem Genet (2013) 51:482–492

123