CLINICAL STUDY: CORONARY ARTERY DISEASE
A common variant of the AMPD1 gene predicts improved cardiovascular survival in patients with coronary artery disease
Jeffrey L. Anderson, MD, FACCa,
Jessica Habashi, BSa,
John F. Carlquist, PhDa,
Joseph B. Muhlestein, MD, FACCa,
Benjamin D. Horne, MPHa,
Tami L. Bair, BSa,
Robert R. Pearson, BSa and
Noal Hart, BSa
a University of Utah, LDS Hospital, Salt Lake City, Utah, USA
Manuscript received December 29, 1999;
revised manuscript received April 3, 2000,
accepted June 2, 2000.
Reprint requests and correspondence: Dr. Jeffrey L. Anderson, University of Utah School of Medicine (4A154), Department of Medicine/Division of Cardiology, 50 North Medical Drive, Salt Lake City, Utah 84132
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Abstract
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OBJECTIVE
We tested whether a common AMPD1 gene variant is associated with improved cardiovascular (CV) survival in patients with coronary artery disease (CAD).
BACKGROUND
Reduced activity of adenosine monophosphate deaminase (AMPD) may increase production of adenosine, a cardioprotective agent. A common, nonsense, point variant of the AMPD1 gene (C34T) results in enzymatic inactivity and has been associated with prolonged survival in heart failure.
METHODS
Blood was collected from 367 patients undergoing coronary angiography. Genotyping was done by polymerase chain reaction amplification and restriction enzyme digestion, resulting in allele-specific fragments. Coronary artery disease was defined as  70% stenosis of 1 coronary artery. Patients were followed prospectively for up to 4.8 years. Survival statistics compared hetero- (+/–) or homozygotic (–/–) carriers with noncarriers.
RESULTS
Patients were 66 ± 10 years old; 79% were men; 22.6% were heterozygous and 1.9% homozygous for the variant AMPD1(–) allele. During a mean of 3.5 ± 1.0 years, 52 patients (14.2%) died, 37 (10.1%) of CV causes. Cardiovascular mortality was 4.4% (4/90) in AMPD1(–) allele carriers compared with 11.9% (33/277) in noncarriers (p = 0.046). In multiple variable regression analysis, only age (hazard ratio, 1.11/year, p < 0.001) and AMPD1(–) carriage (hazard ratio, 0.36, p = 0.053) were independent predictors of CV mortality.
CONCLUSIONS
Carriage of a common variant of the AMPD1 gene was associated with improved CV survival in patients with angiographically documented CAD. The dysfunctional AMPD1(–) allele may lead to increased cardiac adenosine and increased cardioprotection during ischemic events. Adenosine monophosphate deaminase-1 genotyping should be further explored in CAD for prognostic, mechanistic and therapeutic insights.
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Abbreviations and Acronyms
| | AMISTAD | = Acute Myocardial Infarction Study of Adenosine | | AMPD | = adenosine monophosphate deaminase | | CAD | = coronary artery disease | | CV | = cardiovascular | | HR | = hazard ratio | | MI | = myocardial infarction |
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The adenosine monophosphate deaminase-1 (AMPD1) gene encodes an isoform of AMP deaminase (AMPD1, also called myoadenylate deaminase) that is active in muscular tissue (1). Adenosine monophosphate deaminase-1 occupies a central position in adenosine nucleotide catabolism, catalyzing the conversion of AMP to inosine monophosphate, the rate-limiting step for entry into the purine nucleotide cycle. Adenosine monophosphate deaminase-1 deficiency is believed to cause exercise-induced myalgias and early fatigue in skeletal muscle (1–3). A common polymorphism in axon 2 of AMPD1, present in about 25% of Caucasians, causes a C to T transition at nucleotide 34 (C34T) (2,3). This nonsense transition encodes for a truncated, inactive enzyme. A reduced activity of AMPD1 may increase persistence of adenosine (3,4), a cardioprotective molecule (5). Recently, the C34T variant of AMPD1 has been reported to be associated with prolonged survival in heart failure (6,7). We tested whether it also is more broadly associated with improved cardiovascular (CV) survival in patients with coronary artery disease (CAD) at high risk for future ischemic events.
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Methods
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Study objectives.
We tested whether carriage of the common variant allele of the AMPD1 gene, (AMPD1[–]) was associated with a reduced risk of CV death in patients with documented CAD. We also tested its association with all-cause mortality.
Study population.
Study subjects came from a consecutive series of clinically stable patients of any age and either gender who underwent coronary angiography, were shown to have severe CAD, consented for a blood draw at the time of angiography (for confidential blood bank studies approved by the hospital’s institutional review board) and were followed until death or for >2.5 years from entry. Subjects were primarily residents of Utah, a population ethnically primarily of Northern European descent and genetically similar to the general U.S. Caucasian population (8).
Data collection.
At the time of angiography, key demographic characteristics were recorded on standard data forms, including age, gender and history of recent or remote myocardial infarction (MI) (9). Determination of presence and severity of CAD was made by each patient’s attending cardiologist, who was unaware of AMPD1 genotypes, using a format modified after the Coronary Artery Surgery Study protocol (9,10). Severe CAD was defined as the presence of 1 coronary lesions of 70% diameter stenosis in 1 major coronary artery or its primary branch. Mild or absent CAD cases were excluded from this study of secondary risk. Index angiography occurred between August 1994 and December 1997.
Assessment of patient outcomes.
The study patient cohort was followed until death or December 1998 (mean, 3.5 ± 1.0 years of follow-up, range 2.5 to 4.8 years). Each subject was interviewed through a telephone survey that determined the subject’s medical history since the index hospitalization. Deaths were determined when possible from a family member. Deaths were verified and other deaths determined as of March 1999 by a search of a national Social Security database. Subjects unable to be contacted by telephone but not listed as deceased by the national database were considered to be alive. Follow-up using the database allowed for 100% assessment of survival within the cohort.
DNA extraction.
Approximately 20 ml to 30 ml of blood was withdrawn by venipuncture at the time of coronary angiography, collected in ethylenediaminetetraacetic acid, refrigerated at 4°C and processed within 24 h. The leukocyte buffy coat was separated by centrifugation, and genomic DNA was extracted using a standard phenol:chloroform method as previously described (11).
DNA genotyping.
To identify the AMPD1 C34T variant genotypes, polymerase chain reaction amplification was performed with the following primers, as previously published (12):
Amplification reactions were performed in 15 µl volumes containing the two primers. Genotyping was performed as previously reported (13). The reaction products were visualized by electrophoresis through a 2% agarose gel containing ethidium bromide.
Statistical considerations.
Comparisons of characteristics of survivors and nonsurvivors used chi-square (categorical variables) or unpaired t testing (continuous variables). Allelic and genotypic frequencies were determined from observed counts. Comparisons between allelic or genotypic frequency distributions used chi-square analysis. Hetero- (+/–) or homozygotic (–/–) carriers were compared with noncarriers (wild type genotype, [+/+]) using survival statistics. The univariate predictive value of AMPD1(–) carriage for CV and total survival was tested using Kaplan-Meier analysis and log-rank statistics. Cox logistic regression analysis (stepwise, backward logistic regression approach) was then used to determine univariate and multiple variable hazard ratios (HR) and the multiple variable predictive value of AMPD1(–) carriage, conditioned on 10 other major CAD risk factors: age, gender, smoking status, diabetic status, history of hypertension, history of hyperlipidemia, family history, renal failure, presentation and initial therapy (SPSS v 9.0, Chicago, Illinois). The critical value for entering and excluding variables in the model was set at p = 0.10.
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Results
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Baseline patient characteristics.
A total of 367 patients with documented CAD was entered, and 52 patients (14.2%) died during the mean of 3.5 ± 1.0 year follow-up, 37 (10.1%) of CV causes. Selected patient characteristics at study entry are summarized in Table 1 by survival status. Patients averaged 66 ± 10 years of age, and 79% were men. Survivors were younger at baseline, had higher ejection fractions and tended to be men and smokers more frequently than nonsurvivors.
Adenosine monophosphate deaminase-1 genotypic distributions are shown in Table 2 for all patients and for survivors, those dying of any cause and those dying of a CV cause. Of entered patients, 22.6% were heterozygous and 1.9% homozygous for the AMPD1(–) allele. Thus, 24.5% were carriers of the polymorphic allele. In bivariate correlation analyses, AMPD1(–) carriage was unassociated with any other baseline factor, including ejection fraction.
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Table 2 Genotypic Distributions and Allelic Frequencies of AMPD1 Gene Polymorphism Among Patients by Survival Status
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AMPD1 genotype and survival.
At the end of follow-up, CV mortality was 4.4% (4/90) for AMPD1(–) allele carriers compared with 11.9% (33/277) for noncarriers. Figure 1 shows the time-to-event (Kaplan-Meier) CV survival plot as a function of AMPD1(–) allele carriage. A significant difference in survival by AMPD1 genotype was observed (log-rank statistic, 4.0, p = 0.046). The HR of death for AMPD1(–) carriage was 0.36 (0.13 to 1.0).
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Figure 1 Kaplan-Meier survival-time plot for cardiovascular death by AMPD1 genotype (wild type vs. variant heterozygote or homozygote). There are four events (4.4%) among 90 patients carrying the mutant polymorphism and 33 events (11.9%) among noncarriers, a statistically significant difference (log-rank statistic 4.0, p = 0.046; Breslow statistic 4.1, p = 0.043). Solid line = AMPD1(–) variant carrier; dotted line = variant noncarrier.
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Adenosine monophosphate deaminase-1(–) carriage was not associated with a reduction in noncardiovascular deaths (5.5% in carriers, 3.6% in noncarriers). When all-cause mortality was considered (CV plus non-CV), the difference was not significant (10.0% in AMPD1[–] carriers, 15.5% in noncarriers; p = 0.19).
In multiple variable Cox regression analysis, including 12 clinical and laboratory variables (age, gender, smoking, diabetes, hypertension, hyperlipidemia, family history, total cholesterol, renal failure, presenting diagnosis, therapy at index hospitalization and AMPD1[–] carriage), only age (HR, 1.11/year, p < 0.001) and AMPD1(–) carriage (HR, 0.36, confidence interval 0.13–1.02, Wald chi-square p = 0.053) were selected as independent predictors of CV mortality (Table 3).
The incomplete database for ejection fraction dissuaded us from doing a formal determination of the relative predictive value of the polymorphism in high and low ejection fraction subgroups. However, the reduction in CV mortality did appear to be prominent in those with low ( 40%) ejection fractions (0/9 with, vs. 13/34 without an AMPD1[–] allele and documented low ejection fraction, died).
In contrast with its value for secondary risk prediction, AMPD1 polymorphism was not useful for prediction of the presence or absence of CAD at initial angiography in an expanded consecutive series that included subjects with normal angiograms (not shown).
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Discussion
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Study summary.
We found that patients with angiographically documented CAD who were carriers of a common genetic variant of the AMPD1 gene demonstrated improved CV survival. The AMPD1 variant did not predict development of CAD; rather, the effect appeared to be in prolonging survival when heart disease was already present. Adenosine monophosphate deaminase-1 genotype was unassociated with other risk factors, and its predictive value was undiminished in multiple variable analyses (HR = 0.36). We speculate that the dysfunctional AMPD1(–) allele may lead to increased net production of adenosine locally (in cardiac muscle [7]) and/or systemically (skeletal muscle source [6]), affording increased levels of cardioprotection during ischemic events. If these results are verified, AMPD1 genotyping may provide useful prognostic, mechanistic and therapeutic insights into CAD progression and prognosis.
Previous work.
Recently, Loh et al. (6) reported an improved clinical outcome associated with AMPD1(–) allele carriage in a group of 132 patients with advanced heart failure referred for cardiac transplant evaluation. The mutant AMPD1 allele was associated with an extended time from the first hospitalization for heart failure to evaluation for transplantation, with an HR for transplant-free survival of 4.6. Our study is the first to confirm and extend these findings to patients with CAD who were not selected by ejection fraction or heart failure and who were studied prospectively after angiographic diagnosis.
Mechanisms of benefit.
Loh et al. (6) speculated that the mechanism of benefit could be related to enhanced production of adenosine in skeletal muscle that could increase circulating levels of adenosine, leading to cardioprotection. Feldman et al. (7) editorialized that the short circulating half-life of adenosine argued for a primarily local (myocardial) increase in net adenosine and hypothesized that adenosine levels might be increased in cardiac muscle in patients carrying the variant allele (7). Adenosine, released by myocytes during ischemic stress (13), has been studied extensively for a cardioprotective role although this remains to be completely defined (14,15). As reviewed by Mahaffey et al. (15), adenosine has been reported to replenish high-energy phosphates, inhibit oxygen free radical formation and neutrophil activation and accumulation, improve microvascular function and participate in myocardial ischemic preconditioning in experimental models of occlusion/reperfusion, improving cardiac perfusion and function.
Earlier human studies (16,17), promising in themselves, have been followed by a larger (n = 236 patients) controlled study, the Acute Myocardial Infarction Study of Adenosine (AMISTAD) (15). In AMISTAD, adenosine (70 µg/kg/min) infused for 3 h as an adjunct to thrombolytic therapy reduced radionuclide infarct size by 33% (p = 0.03). A larger trial to assess clinical events was proposed.
The role of AMPD1 in cardiac muscle is less well studied than in skeletal muscle although it has been reported to be expressed (together with AMPD2) in mammalian heart (18). To date, neither myocardial nor skeletal muscle adenosine levels have been measured in disease states and by AMPD1 genotype.
Whatever the precise mechanism of adenosine’s benefit, the AMPD1(–) allele may provide carriers with an endogenous source of increased myocardial adenosine, improving outcomes in those with CAD at high risk for future ischemic events.
Study strengths and limitations.
This study extends previous work on clinical consequences of the AMPD1 polymorphism (6) by including a larger and broader spectrum of patients and evaluating their clinical course entirely prospectively. Adenosine monophosphate deaminase-1 genotype was unassociated with other risk factors, and its association with CV survival was independent of other tested risk factors in multiple variable analysis. However, the study is only moderate in size, and the number of clinical events is relatively small, so that the confidence intervals for CV survival extension associated with AMPD1(–) are broad. Similarly, the database for determining the relative protective effect of the variant as a function of ejection fraction is limited. Also, the study did not directly assess potential mechanisms of apparent benefit. The similarity of genotypic frequencies in our CAD group to that in the general population further supports our observation about the absence of an effect on the development of CAD. Thus, for future studies disease progression or prognosis may be a better focus than disease development. In conclusion, our findings, although promising, should be verified and extended in larger and longer-term studies. If validated, they suggest that AMPD1 genotyping may provide useful prognostic, mechanistic and therapeutic insights into survival in patients with CAD as well as those with congestive heart failure.
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Footnotes
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Supported, in part, by grants from the Deseret Foundation, Intermountain Health Care, Salt Lake City, Utah.
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References
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Abstract
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