The clinical significance of a common, functional, X-linked angiotensin II type 2-receptor gene polymorphism (1332 G/A) in a cohort of 509 families with premature coronary artery disease
Khaled Alfakih1,
Richard A. Lawrance1,
Azhar Maqbool2,
Kevin Walters1,
Stephen G. Ball1,
Anthony J. Balmforth2 and
Alistair S. Hall1,*
1BHF Heart Research Centre (Clinical), G Floor, Jubilee Wing, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK
2BHF Heart Research Centre (Laboratory), Jubilee Wing, University of Leeds, Great George Street, Leeds LS2 9JT, UK
Received 20 March 2004; revised 12 September 2004; accepted 16 September 2004; online publish-ahead-of-print 1 December 2004.
* Corresponding author. Tel: +44 113 392 5393; fax: +44 113 392 5405. E-mail address: cvsash{at}leeds.ac.uk
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Abstract
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Aims To assess, in families with premature coronary artery disease (CAD), the possible association, with linkage, of the X-linked AT2 receptor (1332 G/A) gene polymorphism and premature CAD.
Methods and results We investigated 509 families with a history of premature CAD that consisted of one sibling affected with premature CAD and two unaffected siblings. Genotyping of subjects was performed using a restriction enzyme digestion of an initial 310 bp polymerase chain reaction fragment that included the AT2 (1332 G/A) locus. The mean age of the 611 individuals affected by premature CAD at the time of event was 49.5±8.1 years. Conditional logistic regression analysis confirmed a significant predictive value of premature CAD for the covariates of hypertension, diabetes, dyslipidaemia, history of smoking, and male gender. The genetic data were analysed for these families using the X-linked sibling transmission/deletion test (XS-TDT) statistics program. In hemizygous men we observed evidence for association in the presence of linkage, for the AT2 (1332 G/A) locus and premature CAD (P-exact value=0.024) and also a trend towards association, in the presence of linkage, for this polymorphism and hypertension (P-exact value=0.08).
Conclusions We have observed evidence of association between the presence of linkage for the X-linked AT2 (1332 G/A) polymorphism and premature CAD in hemizygous males.
Key Words: Angiotensin Genetics Coronary disease Molecular biology
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Introduction
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A recent genome-wide search conducted in a genetically isolated population in Finland demonstrated linkage between premature coronary artery disease (CAD) and a region on chromosome Xq2326.1 They noted that the angiotensin II type two (AT2) receptor gene is located at the same locus (Xq2326). The authors highlighted the fact that men hemizygotic for disease alleles on chromosome X would have a higher probability of showing the effects of a recessively acting X-chromosome susceptibility gene, than would dizygous women. Consequently, point mutations in this chromosomal region may explain the higher frequency of premature CAD in men compared with women. AT2 receptor agonism is reported to activate matrix metalloproteinases (MMPs) 1 and 9,2 inhibit vascular cell proliferation,3 ameliorate the inflammatory response in vascular injury,4 and regulate collagen-producing cells.5 These represent potential mechanisms by which a functional AT2 receptor gene polymorphism may contribute to the development of disease.
The structure and nucleotide sequence of the AT2 receptor gene in humans was described in 1995.6 It consists of three exons and two introns, with the entire coding region located on exon 3. Recently, a commonly occurring AT2 receptor intronic polymorphism has been reported;7,8 it is located within intron 1 at a lariat branchpoint, 29 bp before the start of exon 2, close to a region that is important for transcriptional activity.9 Its position, described relative to the translation initiation site of the human AT2 receptor gene, is (1332),7 although it has also been described by others as (+1675).8 In this study we have used the former nomenclature. Human mRNA studies confirm differently spliced AT2 human mRNA species and show subjects with the G allele to have exon 2 missing, and the amount of this abnormally spliced mRNA to be markedly reduced. This confirms the functional significance of this point mutation.7 The G allele has already been associated with congenital anomalies of the kidney and urinary tract in men.7 The A allele was reported to be associated with elevated left ventricular mass (LVM), as measured by echocardiography in a small cohort of young mildly hypertensive males,10 and also with a history of ischaemic heart disease in females, in a subgroup of a cohort originally used to investigate the prevalence of left ventricular dysfunction in a random sample of subjects.11 We recently observed a significant association between the AT2 receptor (1332 G) genotype and elevated LVM measured by cardiac magnetic resonance imaging (MRI) in patients with hypertension.12
We wished to evaluate the common and functional X-linked AT2 receptor gene (1332 G/A) polymorphism in a national registry of families with history of premature CAD: the Genetic Risk of Acute Coronary Events (GRACE) cohort.13 This is a genomic DNA library derived from sibling trios with at least one sibling affected with premature CAD and additional unaffected siblings. The size of the cohort is sufficiently large to permit reliable determination of the presence or absence of association in the presence of linkage for the AT2 (1332 G/A) polymorphism and premature CAD. The structure of the families was specifically chosen to permit assessment of transmission disequilibrium of genetic loci. For this study we used the X-linked sibling transmission/disequilibrium test (XS-TDT), which allows the calculation of exact P-values where the null hypothesis is of no linkage. Under certain conditions relating to the number of affected and unaffected siblings, the XS-TDT can be used as a test of association. The XS-TDT requires no parental data and is therefore well suited to diseases of late onset.14 In comparison with the use of unrelated cases and controls, the use of sibling trios avoids the potentially confounding effect of genetic heterogeneity that would tend to result in a false positive finding. In contrast, the use of sibling trios would be predicted to reduce markedly the likelihood of false positive findings due to the presence of much greater genetic homogeneity as a result of shared inheritance.
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Methods
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The GRACE study
The subjects for this study were from the GRACE cohort, a national registry of families with history of premature CAD from whom DNA has been obtained and archived. The GRACE cohort was established to investigate the association between candidate genetic polymorphisms and premature CAD in a large cohort of patients, using unaffected siblings as controls, with rigorous phenotypic assessment. The study recruited families with three siblings, one affected with premature coronary artery disease prior to or at the age of 65 years and two unaffected siblings. The age of 65 years used to define premature is in line with UK statistics that use the age of retirement as a cut-off. The cohort also included 44 families with two siblings (one affected and one unaffected) and 50 families with more than three siblings (at least one affected sibling). The affected individuals had either had a myocardial infarct (MI), confirmed angina, percutaneous coronary intervention (PCI), or coronary artery bypass grafting (CABG) prior to or at the age of 65 years. Written informed consent was obtained from all subjects and the local ethics committee approved the study.
Recruitment
The study and its aims were publicized in a recruitment campaign which involved a bus tour of UK cities as well as television and newspaper interviews. Ten per cent of the subjects who contacted the research group proved to be suitable for the project. Information was sent to the proband and his/her siblings. Three to 4 weeks later the siblings were contacted and a time for interview and phlebotomy was arranged. This was either performed at Leeds General Infirmary or the clinical information was recorded through telephone interview, with the blood sample being taken by the family doctor and posted to Leeds General Infirmary. The clinical information of the patients and their siblings, and dates of such history, were validated by each person's family doctor, based on hospital letters archived by them. In contentious cases, family doctors were recontacted for further information or hospital notes were reviewed. Such cases were not validated unless hospital records confirmed (by angiography of a clearly positive stress test) that CAD was present. This investigation was carried out on the first 509 families to be recruited into the GRACE cohort.
Laboratory methods
Genomic DNA was extracted from peripheral blood using a Puregene kit (Gentra Systems, MN, USA). Primers 5' AGA GAT CTG GTG CTA T TA CG 3' and 5' CAC T TG AAG ACT TAC TGG T TG 3' (Invitrogen) were used to amplify a 310 bp DNA fragment between intron 1 and exon 2 including the A/G polymorphism. Polymerase chain reactions were run at 95°C for 15 min to activate the AmpliTaq Gold enzyme (Perkin-Elmer Applied Biosystems, CA, USA), followed by 35 cycles of: 95°C for 30 s, 42°C for 30 s, and 72°C for 45 s. Subsequently, reactions were run to completion by a final 10 min at 72°C. The product (5 µL) was digested for >3 h with 5 U of the restriction enzyme HYP 188 III (New England BioLabs, MA, USA), which cuts the G but not the A allele. Samples were then subjected to gel electrophoresis (2% agarose) to permit genotyping (Figure 1). The AT2 receptor G allele yields two fragments, of 104 and 206 bp, and the AT2 receptor A allele yields a single undigested 310 bp fragment. Heterozygous females demonstrated all three fragments. Genotyping was found to be accurate when confirmed by direct DNA sequencing (BigDye® Terminator v3.1 Cycle Sequencing Kit; ABI PRISM, 3100 Genetic Analyser, Perkin-Elmer Applied Biosystems, CA, USA) of the first 35 patients (Figure 1).


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Figure 1 Photograph of (A) agarose gel following electrophoresis and ethidium bromide staining of the products of restriction enzyme digestion; lane 1, 100 bp ladder; lane 2, undigested 310 bp fragments (A allele); lanes 35, digested 206 bp fragments (G allele); lanes 67, heterozygous females showing both bands. (B) Electropherogram showing three sequenced patients illustrating the three different genotypes. The second electropherogram represents a heterozygous female where R denotes the presence of both an A and G nucleotide bases at position 1332 of the PCR product.
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Statistical methods
Means and standard deviations were calculated for the age of the cohort and the distribution of the genotype was calculated. The number of validated events and the number of cardiovascular risk factors for the affected individuals were calculated. Conditional logistic regression analysis was performed to assess the effect of hypertension, diabetes, dyslipidaemia, and history of current or previous smoking as well as male gender on the affected versus the unaffected status. An initial univariate conditional logistic regression analysis showed all variables to be significant at the 5% level. Variables were ranked according to the univariate odds ratios. Covariates were considered sequentially for inclusion using a likelihood ratio test (STATA software, version 8, STATA Corporation, TX, USA). The genetic results were analysed using the XS-TDT program.14 This performs the XS-TDT analysis, which gives a P-value where the null hypothesis is of no linkage. A 5% level of significance was used.
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Results
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The mean age for the entire cohort (n=1556) was 57.0±9.0 years of whom 857 (55.1%) were male (mean age 56.7±8.9 years) and 699 (44.9%) were female (mean age 57.2±9.2 years). There were 611 (39.3%) individuals affected by premature CAD with 459 (75.1%) male, mean age 58.0±8.0 years and 152 (24.9%) female, mean age 58.7±8.2 years. The mean age at time of first event for the affected individuals was 49.5±8.1 years. There were 945 (60.7%) unaffected individuals (mean age 56.2±9.5 years), up to the time of study, with 398 (42.1%) male, mean age 55.3±9.6 years and 547 (57.9%) females, mean age 56.8±9.5 years. The distribution of the genotype for the men was: A genotype=446 (52.0%), G genotype=411 (48.0%). For the females, the distribution of the genotype was: AA genotype=186 (26.6%), GA genotype=352 (50.4%), and GG genotype=161 (23.0%).
The distribution of the (1332 G/A) AT2 receptor gene polymorphism in the general population has not yet been established. Consequently, we observed the distribution in a population with no known cardiovascular disease by excluding subjects with a history of premature CAD or history of hypertension from the GRACE cohort (n=702). The distribution of genes in females was AA=109 (27.7%), GA=183 (46.6%), GG=101 (25.7%) and in males was A=180 (58.3%) and G=129 (41.7%). The distribution of the gene for combined homozygous females plus hemizygous males, with heterozygous females excluded (n=519) was A/AA=289 (55.7%) and G/GG=230 (44.3%).
Of the individuals who were affected by premature CAD 515 (84.3%) had definite angina, 470 (76.9%) had an MI, 201 (32.9%) had PCI, and 306 (50.1%) had CABG. Sixty-five (10.6%) of the affected individuals had one validated event, 252 (41.2%) had two validated events, 237 (38.8%) individuals had three validated events, and 54 (8.8%) had four validated events, prior to or at age 65 years. All unaffected siblings were confirmed not to have had any of these events prior to the time of study. Cardiovascular risk factors were recorded for all individuals in the cohort (Table 1).
Conditional logistic regression was used to assess the effect of various cardiovascular risk factors on the affected versus the unaffected status. Hypertension, diabetes, dyslipidaemia, history of current or previous smoking, and male gender were all significant in explaining affected status (P-values <0.005). The odds ratio was highest for dyslipidaemia (Table 2).
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Table 2 The results of the conditional logistic regression analysis, assessing the predictive value of five cardiovascular risk factors with regard to the occurrence of premature CAD within the cohort
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The distribution of the genotype in the males who were unaffected by premature CAD was A=219 (55.0%); G=179 (45.0%) while the distribution of the genotype amongst the males with premature CAD was A=227 (49.5%); G=232 (50.5%). The distribution of the genotype amongst the unaffected females was AA=150 (27.4%); GA=264 (48.3%); GG=133 (24.3%) while the distribution of the genotype amongst females with premature CAD was AA=36 (23.7%); GA=88 (57.9%); GG=28 (18.4%) (Figure 2). The combined family data were analysed using the XS-TDT program,14 which gives a P-value where the hypothesis is of no association and no linkage between genotype and disease state. The G allele occurred significantly more frequently in males with premature CAD than would be expected if the disease susceptible locus and typed marker were unlinked (P-exact value=0.024). The P-exact value for females was 0.939.

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Fig. 2 Bar chart illustrating the increase in frequency of the G allele in male but not female subjects with premature CAD. No-CAD males, A (n=219) 55.0%, G (n=179) 45.0%; CAD males, A (n=227) 49.5%, G (n=232) 50.5%; No-CAD females, AA (n=150) 27.4%, GA (n=264) 48.3%, GG (n=133) 24.3%; CAD females, AA (n=36) 23.7%, GA=(n=88) 57.9%, GG (n=28) 18.4%.
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We recently demonstrated an association between the AT2 receptor gene polymorphism and elevated LVM, as measured by cardiac MRI, in an entirely separate cohort of patients with hypertension.12 This might be explained by a direct effect on the myocardium or alternatively by an indirect effect on blood pressure. To explore the second of these possibilities, we further analysed the GRACE dataset. The distribution of the genotype in the males without history of hypertension was A=312 (54.3%), G=263 (45.7%) vs. A=127 (47%), G=143 (53%) amongst subjects with history of hypertension. For the females, the distribution of the genotype amongst those without history of hypertension was AA=127 (26.9%); GA=231 (48.9%); GG=114 (24.2%) vs. AA=56 (25.6%); GA=120 (54.8%); GG=43 (19.6%) amongst subjects with history of hypertension (Figure 3). Using the XS-TDT program14 to evaluate an association, in the presence of linkage, for the AT2 (1332 G/A) and a history of hypertension, we observed that the G allele occurred more frequently in the hemizygous males (P-exact=0.08) but not dizygous females (P-exact=0.185).

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Fig. 3 Bar chart illustrating the increase in frequency of the G allele in male but not female subjects with a history of hypertension. Normotensive males, A (n=312) 54.3%, G (n=263) 45.7%; hypertensive males, A (n=127) 47.0%, G (n=143) 53.0%; normotensive females, AA (n=127) 26.9%, GA (n=231) 48.9%, GG (n=114) 24.2%; hypertensive females, AA (n=56) 25.6%, GA (n=120) 54.8%, GG (n=43) 19.6%.
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Discussion
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The primary results of this investigation indicate that there is association in the presence of linkage for the AT2 receptor (1332 G/A) polymorphism and premature CAD in men. We found no evidence that this is the case in women. Both premature CAD and hypertension are complex disorders with multiple environmental and molecular risk factors that may affect men and women differently. Hemizygotic men are more likely to show the effects of a recessively acting X-chromosome susceptibility gene than are women. Hence point mutations in the AT2 receptor gene resulting in reduced, but not absent, mRNA formation7 would be predicted to affect men more than it would affect women. However, the smaller number of affected women in the study may have also contributed to the negative result in women. Subjects in this study were from the GRACE cohort, a UK national registry of families with a history of one sibling being affected by premature CAD. The unaffected siblings were confirmed as such through their family doctor up to the time of enrolment in the study. We do not know that the unaffected siblings will remain unaffected until the age of 65 years. However, the mean age of the unaffected individuals when studied was 56.2±9.5 years while the mean age of the affected individuals at time of event was 49.5±8.1 years. The net effect of misclassification would be to make the two populations more similar, i.e. to reduce the sensitivity and increase specificity of any changes. The use of sibling trios avoids the potentially confounding effect of genetic heterogeneity that predisposes to false positive findings in casecontrol studies of unrelated individuals.15
The recent finding of an association between premature CAD and the corresponding region of the AT2 receptor gene on chromosome Xq2326 stimulated us to perform this investigation and is consistent with our results.1 Differently spliced AT2 mRNA species resulting from the polymorphism have been described and human mRNA studies confirm that subjects with the G allele have exon 2 missing and markedly reduced mRNA, which confirms the functional significance of this point mutation.7 Other mutations located in non-coding regions, such as those affecting 5' and 3' splice sites or branch sites can cause hereditary disease.16 Phenotypic variability, as well as variable levels of correctly spliced transcripts, were found among patients carrying such mutations.1719 Inverse correlation between the level of correctly spliced transcripts and disease severity has also been described.20 We have now observed an association, in the presence of linkage, for the AT2 receptor polymorphism (1332 G) and premature CAD in hemizygous males. Furthermore, there was a non-significant trend in the test for association, in the presence of linkage, for the G allele and a history of hypertension in males. Given the size of the cohort and the use of sibling trios, which avoids the potentially confounding effect of genetic heterogeneity, we consider these findings to be robust and clinically relevant. Biological plausibility is further emphasized by consideration of the known effects mediated via the AT2 receptor.
The described role of the AT2 receptor in the regulation of collagen producing cells and hence the structural integrity of atheromatous plaques provides a potential mechanism by which disease might result.5 Also the anti-proliferative actions of the AT2 receptor offset the growth promoting effects mediated by the angiotensin II type 1 (AT1) receptor.3 Angiotensin II (Ang II) is known to induce the release of MMP1 and MMP2, an effect which may predispose to CAD but which can be antagonized by AT1 receptor blockers.21,22 Nevertheless, AT2 receptor-mediated release of MMP1 and MMP9 by macrophages2 may represent a key factor in predisposing to atheromatous plaque fragility and hence rupture. Furthermore, stimulation of the AT2 receptor after AT1 receptor blockade reduces the inflammatory vascular injury.4
Other workers have previously studied the AT2 (1332 A/G) polymorphism in a range of other patient groups. Initially the AT2 (1332 G) allele was reported as showing an association with congenital anomalies of the kidney and urinary tract in a small study of males only.7 Schmieder et al.10 investigated LVM in 120 healthy students (all male) with normal or mildly elevated BP, and reported an association with the AT2 receptor (1332 A/G) polymorphism. The overall allele frequency for this study of relatively unselected individuals was comparable to our own (A=57%; G=43%). Furthermore, in keeping with our own findings, an excess of the G allele was seen in students with a casual BP greater than 140/90 mmHg (G=46%) compared with those deemed to be normotensive (G=41%). It is paradoxical therefore, that students with the A genotype were reported to have slightly higher LVM (as measured by M-mode echocardiography). This contradiction may have resulted from the imprecision of M-mode echocardiography for estimating LVM as it is dependent on geometric assumptions and a mathematical formula with a cube function, which amplifies errors of measurement, resulting in significant variations of LVM estimates. In keeping with the supposition that the G allele is associated with cardiovascular disease, we have previously demonstrated a statistically strong association with LVM as measured much more accurately by 3-D cardiac MRI, for patients with established systemic hypertension.12
Herrmann et al.11 investigated the AT2 receptor gene (1332 G/A) polymorphism and observed an increase in the frequency of the G allele in men with ischaemic heart disease. However, they also reported increased frequency of the A allele in a subgroup of females with a history of ischaemic heart disease. This finding was dependent on the inclusion of heterozygous females, despite the polymorphism being X-linked. In contrast to our own prospective investigation, Hermann's study populations were originally recruited to investigate the prevalence of left ventricular dysfunction.23 As the results were not consistent across the cohort they recommended further research.11 Until now the only other related data were again based on analysis of 322 patients that represent a retrospectively defined subgroup (with systolic, but not diastolic hypertension) taken from a study with a total of 2579 relatively unselected subjects.24 Once again a tentative association with the AT2 receptor gene (1332 A) polymorphism was reported.
Our primary observation of an association between the AT2 receptor (1332 G) gene polymorphism and premature CAD in men, as well as our earlier observation of a significant association with elevated LVM, might both be mediated by a predisposition to hypertension. Experimental antagonism of the AT2 receptor results in an increase in systolic blood pressure.25 To the contrary, stimulation of the AT2 receptor is associated with a vasodilator cascade involving increased production of bradykinin and nitric oxide.25,26 Furthermore, AT1-mediated pressor effects are abolished in transgenic mice that over-express AT2 receptors.27 These experimental observations indicate a vasodilatory role for the AT2 receptor which opposes the pressor effect of Ang II on the AT1 receptor. One might therefore speculate that the proven reduced transcription of the AT2 receptor gene that results from the (1332 G) polymorphism7 produces clinical effects as a result of reduced AT2 receptor-mediated functions. However, there remains a need for any intermediary biological effects of this gene variant to be adequately studied using in vitro techniques.
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Acknowledgements
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This work was carried out in the British Heart Foundation (BHF) Heart Research Centre at Leeds General Infirmary and University of Leeds. Dr Alfakih and Dr Lawrance were both supported by BHF Research Fellowships.
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