deletion polymorphism in the angiotensin-converting enzyme gene and risk of restenosis after coronary angioplasty

in the angiotensin-converting enzyme gene and risk of restenosis after coronary angioplasty

Insertion/deletion polymorphism

Summary

Introduction

Early restenosis in over 30% of cases limits the benefits of percutaneous transluminal coronary angioplasty (PTCA). The mechanisms that underlie restenosis are uncertain, although experimental evidence suggests that the reninangiotensin system is involved in the vascular response to angioplasty. An insertion(I)/deletion(D) polymorphism in the angiotensin-converting enzyme (ACE) gene, which influences plasma ACE level, has been associated with an increased risk of myocardial infarction in those with the DD

Percutaneous transluminal coronary angioplasties is now a well established treatment for coronary (PTCA) artery disease that equals or exceeds that of coronary artery bypasses in several countries. The main limitation of PTCA is restenosis in 30-40% of patients, typically occurring between 1-3 months.’1 Pathologically, the formation of a neointimal layer from proliferating vascular smooth muscle cells that have migrated from the media, plays an important role in the restenotic process. Many variables have been associated with restenosis;2 a possibility that has not been widely considered is that some subjects may be genetically more predisposed to restenosis. Recently, an insertion (I)/deletion (D) polymorphism in the gene for angiotensin converting enzyme (ACE) has been reported to be associated with the risk of myocardial infarction.3 Patients with the DD genotype were found to be at increased risk. The same genotype is associated with an increase in plasma ACE levels.4 ACE converts angiotensin I to angiotensin II. Plasma ACE is predominantly derived from tissue vascular ACE suggesting that patients with the DD genotype may have higher tissue angiotensin II. Angiotensin II stimulates vascular smooth muscle cell growth in vitro,s and in animal models of angioplasty, prior treatment with ACE inhibitors6 or an angiotensin II antagonist reduce neointimal formation. We considered whether patients with the ACE gene DD genotype were intrinsically at an increased risk of restenosis after PTCA, and determined I/D genotypes in a cohort of patients after successful PTCA.

genotype. To

whether this polymorphism influences the risk of restenosis after PTCA, 233 patients who underwent single-vessel angioplasty in the Subcutaneous Heparin and Angioplasty Restenosis Prevention (SHARP) study were genotyped for the I/D polymorphism and pre-PTCA, postPTCA, and 4-month clinical and quantitative angiographic data were compared in the three genotype groups. The groups, (II 53, ID 117, and DD 63) were well matched for baseline clinical and both pre- and post-PTCA angiographic features. At 4-month follow-up there was no significant difference between the genotype groups with respect to any of the quantitative angiographic criteria of restenosis: minimal luminal diameter at the site of the angioplasty (DD 1·35 [SE 0·10] mm, ID/II 1·43 [0·05] mm, difference -0·08 [95% Cl -0·30 to 0·14]), numbers of subjects with more than 50% diameter stenosis (DD 49%, ID/II 46%, relative risk 1·06 [0·79 to 1·43]), or the number of subjects with more than 50% loss of the acute diameter gain after PTCA (DD 54%, ID/II 43%, 1·26 [0·94 to 1·67]). Likewise, there was no difference in the number of subjects with angina or a positive exercise stress test. We conclude that, in patients undergoing elective PTCA, the I/D polymorphism in the ACE gene does not influence the extent of restenosis, and typing for the polymorphism will not be a useful predictor of risk before the procedure.

investigate

Lancet 1995; 345: 1013-16

Departments of Cardiology (N J Samani FRCP, M Brack MRCP, J Cullen MRCP, D P de Bono FRCP, A H Gershlick FRCP) and Medicine (D S Martin BSc, D Lodwick PhD, J D Swales FRCP), University of Leicester, Leicester, UK; Regional Cardiac Unit, Papworth Hospital, Cambridge, UK (A Chauhan MRCP); and The Cardiothoracic Centre, Liverpool, UK (A Harley FRCP) Correspondence to: Dr N J Samani, Department of Cardiology, Clinical Sciences Wing, Glenfield General Hospital, Leicester LE3 9QP, UK

Patients and methods Patients were from the Subcutaneous Heparin and Angioplasty Restenosis Prevention (SHARP) trial,8 which assessed subcutaneous unfractionated calcium heparin on angiographic restenosis after PTCA. Patients who had undergone successful PTCA (defined as a visually assessed diameter stenosis of less than 50% after PTCA) were randomised to receive no heparin (n=172) or 12 500 IU of heparin subcutaneously (n=167), twice daily for four months. Quantitative coronary angiography was done before PTCA, immediately after PTCA, and when symptoms recurred, or electively at 4 months. 40 patients defaulted and 2 patients died during the trial period. 32 patients had multi-vessel PTCA. Of the remaining 265 patients who had single-vessel PTCA and complete clinical and angiographic follow-up, 233 (88%) consented to providing a blood sample for genotype determination. Identical orthogonal angiograms of the lesion were taken on each occasion and quantitatively analysed9 at one centre by one operator, blinded to patient randomisation. DNA was prepared from whole blood collected in EDTA with a DNA extraction matrix (Instagene, Biorad, UK). Genotypes were determined by PCR amplification of the I/D region of the ACE gene with oligonucleotide primers, resolving the amplified products (I allele 490 bp; D allele 190 bp) on 1-5% agarose gels containing

1013

(%) unless otherwise stated. M!=myocard!a! infarction; RCA=right coronary artery; Data

are no

LAD=left anterior

10 bromide. Genotypes were determined without knowledge of the results of the quantitative angiography. Differences between genotype groups were compared by ANOVA or X2 contingency tables as appropriate, in the case of percentage diameter stenosis by ANOVA after arcsine transformation.

Results The

frequency of genotypes (53 [22’7%] II, 117 [502%] [27-1%] DD) was consistent with the study population being in Hardy-Weinberg equilibrium and similar to those previously described in other populations. To check for misclassification, 20% of subjects were randomly chosen for repeat genotyping; the previously assigned genotype was confirmed in all. The ID, and 63

genotype groups were well matched for age, sex, other cardiovascular risk factors, duration of angina, history of previous myocardial infarction, and proportions of angioplasties carried out in the left anterior descending, circumflex, and right coronary arteries (table 1). The proportion of subjects undergoing PTCA for unstable angina (defined as increased symptoms with episodes of

significantly

in the II group

Pre-PTCA obstruction diameter and percentage diameter stenosis were similar between the groups and there was no significant difference between the groups in

Data are mean (SE). Acute gain=obstruction diameter after angioplasty -obstruction diameter before angioplasty; late loss=obstruction diameter after angioplasty -obstruction diameter at follow-up; *p
Table 2:

1014

Quantitative angiographic findings

I

I

I

40

60

80

I

100

% diameter stenosis

of percentage diameter stenosis before percutaneous transluminal coronary angioplasty (PTCA), immediately after PTCA, and at follow-up in subjects grouped according to ACE I/D genotypes

Figure: Cumulative distribution

ethidium

was

I

20

descending

artery; LCx=left circumflex artery; ACEI=ACE-nhibitor treatment. *Differences between groups p=0.01. Table 1: Characteristics of patients

pain at rest requiring hospital admission) higher in the ID and DD groups than (X2=9’3, p=001).

I

0

either of these values

curves

follow-up (F=0’38, p=0’68 and F=2-27, p=011) (table 2). The difference in obstruction diameter at follow-up between the DD genotype and the II/ID genotypes combined was -0-08 (95% CI -0-30 to 0-14). Post-PTCA obstruction diameter in the ID group was significantly less than in the other groups. However, at

the PTCA vessel diameter in the ID group was also slightly smaller than in the other groups and there was no difference in the percentage diameter stenosis after PTCA between the ID and other groups. The overall outcome (acute gain-late loss) was also not different between the groups (F=0-90, p=0-41, DD vs ID/II, -0-08 mm (-0-27 to 0-10). The cumulative frequency curves of percentage diameter stenosis of the three genotype groups pre-PTCA, post-PTCA, and at follow-up are shown in the figure. There was also no significant difference in the number of subjects with more than 50% loss of the acute gain (=4-24, p=0-12) or more than 50% diameter stenosis at follow-up (X2=2’24, p=0-33) between the genotype groups (table 3). Relative risk of restenosis for DD vs II/ID subjects according to these two criteria for restenosis were 1-26 (95% CI 0-94 to 1-67) and 1-06 (0-79 to 1-43), respectively. Likewise, there was no significant difference between the groups in the numbers of subjects with angina (X2=3’90, p=0’14) or a positive exercise stress test (=0-53, p=0’77) at follow-up. When the data were separately analysed for only those with stable angina, the results were similar. 5, 16, and 11I patients in the II, ID, and DD groups, respectively, required early repeat angioplasty for recurrent symptoms (X2=1’57, p=0-46); exclusion of these subjects from the analysis did not alter the results.

MLD=mmimal luminal diameter; ETT=exercise stress test; *Includes patients requiring early reinvestigation. tPositive ETT was defined as the presence of both chest pain and ST changes (not all patients had an ETT at follow-up). Table 3: Restenosis rates and clinical symptoms at follow-up

with theirs. It is possible that the effect of the genotype varies in different ethnic groups, and in this context it is important to bear in mind that the I/D polymorphism, located in intron 16, is probably not directly responsible had no examined. Heparin treatment, by itself, significant for the effect of the locus but acts as a marker for the true effect on restenosis." Subjects receiving heparin were susceptibility polymorphism (Ss).’8 The relation between equally divided between the ACE genotype groups, and the two polymorphisms could vary in different there was no interaction between heparin treatment and of evaluated on the restenosis variables ACE genotype any populations. Alternatively it is possible that the different results reflected the different contexts in which the (data not shown). PTCAs were undertaken although, again, it is difficult to For the 32 patients who had single-vessel PTCA but from whom DNA was not available, mean age was 54-6 identify a simple mechanism to explain the difference on this basis. obstruction diameter and percentage (SE 1-5) years, 0-81 diameter stenosis pre-PTCA were (0-06) mm and Assuming a within-genotype standard deviation of 72-3 (2-0)% respectively, and at follow-up were 1-45 20%, our study had 80% power at p=0-05 to detect a difference of 8% in percentage diameter stenosis at for all variables (0-14) mm and 49-5 (4-2)% (p>0-05 with recruited follow-up between the DD and II/ID genotype groups. compared group). For the two commonly used categorical definitions of Discussion restenosis, restenosis of more than 50% stenosis at followup and more than 50% loss of the luminal gain, relative Restenosis after PTCA has implications not only for the risks for DD vs ID/11 genotypes observed were 1-06 (95% individual patient but also for cost. Although restenosis is CIs=0-79, 1-43) and 1-26 (0-94, 1-67) respectively, not an all-or-none process, prior identification of those indicating that, although a small effect of the less likely to benefit long term is important, even when the factor involved is not directly amenable to polymorphism on restenosis cannot be excluded, such an effect, if present, would not be clinically manipulation, as alternative treatments, such as bypass useful in determining treatment strategies. Our results, surgery, are available. however, do not exclude the possibility that other Evidence from animal models suggests that the reninpolymorphisms in the ACE gene may prove more strongly angiotensin system is involved in the vascular response to predictive of restenosis, or that the I/D polymorphism balloon injury. ACE has been shown to be induced in may interact with polymorphisms in other genes (eg, in such lesionsand angiotensin II promotes smooth-muscle the angiotensin type 1 receptor) in influencing restenosis cell proliferation in the injured wall. 13 ACE inhibitors6 and risk, as has recently been reported for myocardial more recently angiotensin II receptor antagonists7 have infarction. 19 been shown to significantly reduce the size of the Two other aspects of our study could have introduced neointima. In our study, we explored the possibility that a biases: it was primarily designed to investigate the effect polymorphism at the ACE gene locus on chromosome 17, of heparin treatment and not the ACE genotype on which is associated with higher levels of plasmaand PTCA outcome and recruitment of patients for genetic cellularACE, may define subjects at an increased risk of analysis was retrospective and therefore incomplete. restenosis after PTCA. However, we found no difference However, apart from the number of subjects with in any of the various criteria commonly used to define unstable angina, the three genotype groups were well angiographic restenosis or in clinical outcome in subjects matched for other clinical and angiographic criteria and homozygous for the polymorphism (DD gentoype) there was no interactive effect of genotype and heparin compared with the other genotypes. therapy. Likewise, analysis of the 32 patients in whom Despite positive experimental data, randomised studies DNA was not available show them to be similar to the in human beings have, so far, shown no benefit of ACE recruited group with respect to both pre-PTCA variables inhibition in the prevention of restenosis after PTCA.15,16 as well as outcome. Various explanations, including dosage differences and An intriguing finding was a significant difference species specificity, have been advanced to explain this lack between ACE-genotype groups in the number of cases of effect. Our findings seem to exclude a confounding PTCA for unstable angina. As the undergoing effect of the ACE genotype masking any effect of pathogenesis of unstable angina and acute myocardial treatment; they do not, however, exclude the possibility infarction are believed to be similar20 this finding could be that there may be an interaction between the genotype interpreted as providing support for the previously and response to ACE inhibition. In our study, too few reported association between the ACE DD genotype and subjects were taking ACE inhibitors (table 1) to analyse risk of myocardial infarction.3 However, this finding needs separately. However, similar genetic analysis to that to be received with caution as it comes from post-hoc carried out here of the subjects recruited in the ACE analysis and it is based on small numbers. inhibitor studies’’’-’" may provide an answer to this Because we found no evidence that variation at the question. ACE gene defined by the I/D polymorphism influences Our findings contrast with a Japanese report of 82 extent of restenosis at 4 months, we conclude that the emergency PTCAs for acute myocardial infarction" in determination of ACE I/D genotype is unlikely to be which patients with the DD genotype had a 4-1-fold useful in identifying patients at higher risk of restenosis higher risk of restenosis, defined by a greater than 50% after PTCA. reduction in the luminal diameter of the stenosis at follow-up angiography undertaken between 3 and 6 We thank Dr G Epstein and Sister R Carey for help with the collection months after angioplasty compared with findings of blood samples, Dr J Thompson for statistical advice, and the immediately following PTCA compared with those with cardiologists at the three centres for allowing us to study their patients. the II and ID genotypes. Quantitative angiographic data This study was supported by the British Heart Foundation and the was lacking and it is difficult to directly compare our data Nuffield Foundation. Because

patients in the SHARP trial were randomised receive or not receive heparin, any possible interaction between ACE genotype and heparin treatment was also to

1015

Nobuyoshi M, Kimura T, Nosaka H,

al. Restenosis after successful percutaneous transluminal coronary angioplasty: serial angiographic follow-up of 299 patients. J Am Coll Cardiol 1988; 12: 616-23. 2 Landau C, Lange RA, Hillis LD. Percutaneous transluminal coronary angioplasty. N Engl J Med 1994; 330: 981-93. 3 Cambien F, Poirier O, Lecerf L, et al. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 1992; 359: 641-43. 4 Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I converting gene accounting for half the variance of serum enzyume levels. J Clin Invest 1990; 86: 1343-46. 5 Schelling P, Fischer H, Ganten D. Angiotensin and cell growth: a link to cardiovascular hypertrophy. J Hypertens 1991; 9: 3-15. 1

et

Powel JS, Clozel JP, Muller RKM, et al. Inhibitors of angiotensinconverting enzyme inhibit myointimal proliferation after vascular injury. Science 1989; 245: 186-88. 7 Prescott MF, Webb RL, Reidy MA. Angiotensin converting enzyme inhibitor versus angiotensin II, AT1 receptor antagonist: effects on smooth muscle cell migration and proliferation after balloon catheter injury. Am J Pathol 1991; 139: 1291-96. 8 Brack M, Ray S, Chauhan A, et al for the SHARP investigators. Subcutaneous heparin and angioplasty restenosis prevention: results of a multicentre randomised trial evaluating unfractionated heparin. Br Heart J 1994; 71: P29 (abstr). 9 Alvarez LG, Jackson SA, Berry A, Eichhorn EJ. Evaluation of a personal computer-based quantitative coronary analysis system for rapid assessment of coronary stenosis. Am Heart J 1992; 123: 1500-10.

6

10

P. Statistical methods in medical research. Oxford: Blackwell Scientific Publications, 1971: 356-57. 12 Rakugi H, Kim D-K, Kreiger JE, Wang DS, Dzau VJ, Pratt RE. Induction of angiotensin converting enzyme in the neointima after vascular injury: possible role in restenosis. J Clin Invest 1994; 93: 339-46.

11

References

Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin I converting enzyme gene (DCPI). Nucleic Acid Res 1992; 20: 1433.

Transfer of immunised

Armitage

MJAP, Lombardi DM, Bosman FT, Schwartz SM. Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circ Res 1991; 68: 450-56. Costerousse O, Allegrini J, Lopez M, Alhenc-Gelas F. Angiotensin I converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in T-lymphocytes. Biochem J 1993; 290:

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15 MARCATOR Study Group. Does the new angiotensin converting enzyme inhibitor cilazapril prevent restenosis after percutaneous transluminal coronary angioplasty? Circulation 1992; 86: 100-10. 16 MARCATOR Investigators. Angiotensin converting enzyme inhibition and restenosis: the final results of the MARCATOR trial. Circulation 1992; 86: 153 (abstr). 17 Ohishi M, Fuji K, Minamino T, et al. A potent restenosis. Nature Genetics 1993; 5: 324-25.

18 Cambien

19

F, Costerousse O, Tiret L,

et

al. Plasma level and gene

polymorphism of angiotensin-converting enzyme in relation to myocardial infarction. Circulation 1994; 90: 669-76. Tiret L, Bonnardeaux A, Poirer O, et al. Synergistic effects of angiotensin-converting enzyme and angiotensin-II type 1 receptor gene polymorphisms on risk of myocardial infarction. Lancet 1994; 344: 910-14.

20 Fuster V, Badimon L, Badimon JJ, Chesbro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992; 326: 310-18.

myeloma idiotype-specific immunity from marrow

genetic risk factor for

an

actively

donor

Summary

Introduction

idiotype of myeloma immunoglobulin can be used as a unique tumour-specific antigen. We tested the hypothesis that tumour antigen-specific immunity can be transferred from bone-marrow-transplant donor to recipient. We immunised a healthy sibling donor with myeloma immunoglobulin from the plasma of the recipient, conjugated to an immunogenic carrier protein and emulsified in an adjuvant, before marrow transplantation. Detection of a lymphoproliferative response, a parallel response in the carrier protein, recovery of a recipient CD4+ T-cell line with unique specificity for myeloma idiotype, and demonstration by in-situ hybridisation that the cell line was of donor origin, proved that a myeloma idiotype-specific T-cell response was successfully

The variable regions of immunoglobulin heavy and light chains combine to form the unique antigen-recognition site of antibodies and contain determinants that can themselves be recognised as antigens, or idiotypes. The idiotypic determinants of the immunoglobulin synthesised by a clonal B-cell cancer are unique and can thus serve as a tumour-specific antigen.’ In a pilot study, we observed that purified autologous idiotype protein could be made into an immunogenic vaccine by chemical conjugation to a carrier protein in patients with lymphoma.2

The

transferred to the recipient. Donor immunisation with myeloma idiotype may represent a new strategy for enhancing the specific antitumour effect of allogeneic marrow grafts. Lancet 1995; 345: 1016-20

Biological Response Modifiers Program, National Cancer Institute, Frederick, MD 21702-1201, USA (L W Kwak MD, PhD, P L Duffey RN, C W Reynolds PhD, D L Longo MD); Clinical Immunology Services, Program Resources Inc/DynCorp, Frederick (D D Taub PhD); and Fred Hutchinson Cancer Research Center, Seattle, WA, USA (W I Bensinger MD, E M Bryant PhD)

Correspondence to: 1016

Dr L W Kwak

No curative treatment exists for patients with advanced a median survival of about 30 months has remained constant for the past three decades.3 Allogeneic bone-marrow transplantation provides an opportunity to add the antitumour effects of adoptive immunotherapy to those of high-dose chemotherapy. Antigen-specific immunity to viral antigens can be transferred from healthy immune marrow donors to decrease host susceptibility to infection after transplantation.4-6 However, the transfer of antigenspecific tumour immunity has not been studied previously, because of the lack of a refined tumour antigen that could be safely used to immunise the donor.

multiple myeloma;

We immunised an HLA-matched sibling marrow donor with myeloma IgG isolated from the plasma of the transplant recipient, conjugated to a carrier and emulsified in an adjuvant. Our objective was to induce