Aldehyde dehydrogenase 2 (ALDH2) is capable of

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1 CLINICAL STUDY Impact of Genetic Variation in Aldehyde Dehydrogenase 2 and Alcohol Consumption on Coronary Artery Lesions in Chinese Patients with Stable Coronary Artery Disease Lei Xu, 1, MD, Gang Zhao, 1, MD, Jingfeng Wang, 1, MD, Cheng Shen, 1 MD,XiaoLi, 1 MD, Fei Lu, 2 MD, Hongfa Jiang, 2 MD, George Liu, 3 PhD, Kai Hu, 1 PhD, Yanhua Tang, 2 MD, Aijun Sun, 1,4 MD and Junbo Ge, 1,4 MD Summary The aim of this study is to investigate the impact of aldehyde dehydrogenase 2 (ALDH2) genotype and alcohol consumption on coronary artery lesions in Chinese patients with stable coronary artery disease (CAD). A total of 753 Chinese patients (73.6% male) diagnosed with stable CAD by coronary angiography were included in the sample. The frequency of the mutated type ALDH2 2 (including ALDH2 1/ 2 and ALDH2 2/ 2) was 44.1% in CAD patients. The mutated ALDH2 carriers were more susceptible to multivessel lesions. Low to moderate alcohol consumption is related to higher plasma HDL-C level and fewer coronary artery lesions in CAD patients with ALDH2 wild genotype, while these effects were not observed in CAD patients with ALDH2 mutated genotype. Furthermore, we divided the mutated group into heterozygous and homozygous subgroups, and no obvious relationships were observed among drinking and HDL-C and coronary lesions. To explore the metabolic effects of ALDH2 modified by alcohol, we examined the impact of ALDH2 genotype and alcohol intake on HDL-C levels in ALDH2 wild type and knockout mice. The results showed HDL-C levels were significantly higher post low to moderate alcohol consumption in wild type rather than in knockout mice. CAD patients with mutated ALDH2 genotype are inclined to suffer with coronary artery lesions than wild type subjects. Low to moderate ethanol intake contributes to fewer multivessel lesions in CAD patients with ALDH2 wild type, which might be related to higher HDL-C level. (Int Heart J Advance Publication) Key words: ALDH2 genotype, Alcohol intake, High-density lipoprotein cholesterol Aldehyde dehydrogenase 2 (ALDH2) is capable of converting acetaldehyde into acetate, thus plays a crucial role in the oxidation of acetaldehyde. 1) Carriers of the ALDH2 2 (Glu504Lys) genotype are known to have incompetent enzymatic activity and are susceptible to accumulation of acetaldehyde, especially after alcohol intake. 1) Nearly 40-50% of East Asians carry at least one mutant copy of ALDH2. 2-4) ALDH2 2 exerts a dominant negative effect over wild type ALDH2*1/*1, and reduced ALDH2 activity were found in the heterozygote ALDH2*1/*2 carriers, but not in the homozygote ALDH2 2/ 2 carriers. 2) In the past few years, research results from our group and others indicate that ALDH2, as the key enzyme of the major intra-mitochondrial cardiac protector, plays its cardiovascular protective role mainly through modulating glucose utilization and plasma lipid profile. 5-7) Higher high-density lipoprotein cholesterol (HDL-C) level is cardioprotective and is inversely related to the incidence of coronary artery disease (CAD). 8-10) Results from several epidemiological studies demonstrated that chronic low to moderate alcohol consumption might also have cardiovascular protective effect, partly through its role on increasing HDL-C concentration ) Despite the knowledge of the relationship between alcohol consumption and increased HDL-C level, the underlying mechanism responsible for observed interaction remains elusive. Several studies have hinted that low to moderate ethanol protected the heart from ischemic injury by activating ALDH2. 5,14) ALDH2 inactivation induced dyslipidemia. 15,16) Thus, it is From the 1 Shanghai Institute of Cardiovascular Diseases, Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China, 2 Department of Cardiovascular Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, China, 3 Institute of Cardiovascular Science, Key Laboratory of Molecular Cardiovascular Sciences, Peking University Health Science Center, Beijing, China and 4 Institute of Biomedical Science, Fudan University, Shanghai, China. These authors contributed equally to this work. This work was supported by grants from the National Natural Science Foundation of China ( ) and a grant of Innovative Research Groups of the National Natural Science Foundation of China ( ). Address for correspondence: Aijun Sun, MD, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institute of Biomedical Science, Fudan University, Shanghai , China. sun.aijun@zs-hospital.sh.cn or Yanhua Tang, MD, Department of Cardiovascular Surgery, The Second Affiliated Hospital of Nanchang University, 190 Jiangling West 2nd Rd, Qingyunpu Qu, Nanchang , China. tyh6565@163.com Received for publication March 26, Revised and accepted September 26, Released in advance online on J-STAGE June 6, doi: /ihj All rights reserved by the International Heart Journal Association. 1

2 2 Xu, ET AL IntHeartJ Advance Publication tempting to speculate that the cardioprotective role of low to moderate alcohol consumption may be linked with ALDH2-mediated HDL-C elevation. In the present study, we tested above hypothesis by observing coronary artery lesion severity and HDL-C level of stable CAD patients with various ALDH2 genotypes and the role of alcohol consumption on coronary artery atherosclerosis with various ALDH2 genotypes. To further investigate the relationship of HDL-C levels with the ALDH2 genotype and the impact of alcohol consumption, we also examined the influence of low to moderate alcohol consumption on HDL-C level in ALDH2 wild type mice and ALDH2 knockout mice. Methods Study population: The clinical investigation was conducted in compliance with the guidelines for genetic research and the study protocol was approved by the Ethics Committees of Zhongshan Hospital. The patients suspected of stable angina pectoris were hospitalized for coronary angiography in the Cardiology Department of Zhongshan Hospital from October 2015 to September Those who were suffered with over 75% stenosis of at least one major coronary artery (left main artery, left anterior descending artery, left circumflex artery or right coronary artery) were diagnosed with stable CAD and enrolled into this study consecutively. More than single vessel lesion was identified as multivessel lesions. Clinical information including date of birth, gender, incidence of diabetes mellitus, hypertension and smoking, lipid profiles including levels of HDL-C, low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG), liver and renal function level, and ALDH2 genotype (detected by Laboratory Department of Zhongshan Hospital) were analyzed. Information on alcohol intake: Information on alcohol intake from each subject was obtained using a questionnaire administered by an experienced public health nurse. According to the recommendation for sensible drinking limits made by the Royal College of Physicians, Psychiatrists, and General Practitioners, the total intake of ethanol was converted into standard units (1 unit = 8 g ethanol) per week for each subject. 17) All subjects were then divided into three categories by units of ethanol intake: nondrinker (< 1 unit per week), low to moderate drinker (1-21 units per week for men, or 1-14 units per week for women), and excessive drinker (> 21 units per week for men, or > 14 units per week for women). 17) Animals and ethanol feeding protocols: Animal studies were performed to further investigate the relationship of HDL-C levels with the ALDH2 genotypes post alcohol consumption. All animal experiments were approved by the Animal Care Committee of Zhongshan Hospital, Fudan University. Twenty adult male C57BL/6 mice (8 weeks old, WT group) were obtained from the Shanghai Animal Administration Center (Shanghai, China), and 20 age- and weight-matched ALDH2 knockout mice (KO group) were produced as described previously. 18) All mice were maintained at room temperature under a 12-hour/12- hour light/dark cycles with free access to standard chow and water. The animals were randomly assigned to receive water (n = 10 each genotype) or alcohol (n = 10 each genotype). The ethanol feeding protocol was similar to that described previously (2.5% for the first week, 5% for the second week, 10% for the third week, and 18% for the 4th to 7th week). 5) Lipid analysis in mice: For the lipid analysis, blood samples were collected following an overnight fast. HDL-C was measured and quantified after the precipitation of apolipoprotein B-containing lipoproteins with an equal volume of a 20% polyethylene glycol solution, as described previously. 19) Statistical analysis: The data shown are presented as the mean ± SD. Student s t-test was used for 2-group comparisons and the Chi-square test was used to compare dichotomous variables. Multi-group comparisons were performed by one-way ANOVA with SNK test. Multivariate step-wise logistic regression analyses were used to determine the predictors of multivessel lesions in patients with different genotypes. Hazard ratios were presented with 95% confidence intervals. Variables known or suspected to be associated with outcome were included in the models, involving male gender, age, smoking, hypertension, diabetes, drinking, lipid levels. A two-tailed < 0.05 was considered statistically significant. Results Association between ALDH2 genotype and coronary vessel lesion in CAD patients: A total of 753 subjects [554 men (73.6%), mean age 63.4 ± 10.3 years old] were enrolled. Table I presents data of patients with various ALDH2 genotypes. The frequency of the mutated type ALDH2 2 (including ALDH2 1/ 2 and ALDH2 2/ 2) was 44.1% in stable CAD patients. Gender distribution, age, smoking, alcohol consumption, hypertension, diabetes, TG, HDL-C and LDL-C levels, liver and renal function levels were similar between ALDH2 1/ 1 and ALDH 2 2 groups, however, percent of multivessel lesions was significantly higher in ALDH2 mutant homozygous genotype group than in ALDH2 1/ 1 group. Impact of alcohol consumption on coronary artery lesions in patients with wild and mutated ALDH2 genotypes: Among CAD patients with wild ALDH2 genotype, excessive drinkers tended to be male smokers (Table II). HDL-C level was significantly higher in low to moderate drinking group than in no drinking group (Table II). Percent of multivessel lesions was significantly lower in low to moderate drinking group than in no drinking group and excessive drinking group (Table II). In mutated ALDH2 group, the prevalence of smokers was significantly higher in excessive drinking group, while plasma HDL-C level and percent of multivessel lesions were similar among groups (Table III). The incidence of multivessel lesions among excessive drinkers was a little higher in the group with mutant genotype (39.3%, Table III) than in the group with wildtype (33.3%, Table II). And among subjects with wildtype genotype, the incidence of multivessel lesions in no drinking group (28.9%) was lower than in excessive drinking group (33.3%), though the was no statistical significance (Table II). This may be due in part to the higher prevalence of smoking in the latter (32.9% ver-

3 IntHeartJ Advance Publication ALDH2 AND ALCOHOL CONSUMPTION IN CHINESE CAD 3 Table I. Comparison of Clinical Characteristics in ALDH2 Wild and Mutated Subjects Total (n = 753) ALDH2*1/*1 (n = 421) ALDH2*1/*2 (n = 293) ALDH2*2*2 (n = 39) Males, n (%) 554 (73.6%) 310 (73.6%) 212 (72.4%) 32 (82.1%) Age (years) 63.4 ± ± ± ± Smoking, n (%) 300 (39.8%) 178 (42.3%) 106 (36.2%) 16 (41.0%) Alcohol 0.463, n (%) 543 (72.1%) 301 (71.5%) 215 (73.4%) 27 (69.2%) Low to moderate drinking, n (%) 131 (17.4%) 69 (16.4%) 53 (18.1%) 9 (23.1%) Excessive drinking, n (%) 79 (10.5%) 51 (12.1%) 25 (8.5%) 3 (7.7%) Hypertension, n (%) 217 (28.8%) 123 (29.2%) 79 (27.0%) 15 (38.5%) Diabetes, n (%) 139 (18.5%) 75 (17.8%) 54 (18.4%) 10 (25.6%) TG (mmol/l) 1.76 ± ± ± ± HDL-C (mmol/l) 1.10 ± ± ± ± LDL-C (mmol/l) 2.07 ± ± ± ± ALT (U/L) 35.5 ± ± ± ± Creatinine (μmol/l) 83.3 ± ± ± ± Multivessel lesions, n (%) 225 (29.9%) 112 (26.6%) 96 (32.8%) 17 (43.6%)* Compared with ALDH2*1/*1 group. *P < Table II. Effect of Alcohol Consumption on Coronary Artery Lesions in Patients with Wild ALDH2 Genotype (n = 301) Low to moderate drinking (n = 69) Excessive drinking (n = 51) Males, n (%) 215 (71.4%) 49 (71.0%) 46 (90.2%)* Age (years) 63.3 ± ± ± Smoking, n (%) 99 (32.9%) 32 (46.4%)* 47 (92.2%)* Hypertension, n (%) 84 (27.9%) 26 (37.7%) 13 (25.5%) Diabetes, n (%) 57 (18.9%) 12 (17.4%) 6 (11.8%) TG (mmol/l) 1.83 ± ± 0.97* 1.83 ± HDL-C (mmol/l) 1.09 ± ± 0.39* 1.14 ± LDL-C (mmol/l) 2.05 ± ± ± ALT (U/L) 35.8 ± ± ± Creatinine (μmol/l) 82.6 ± ± ± Multivessel lesions, n (%) 87 (28.9%) 8 (11.6%)* 17 (33.3%) Compared with group, *P < 0.05; compared with Low to moderate drinking group, P < 0.05). Table III. Effect of Alcohol Consumption on Coronary Artery Lesions in Patients with ALDH2 Mutated Genotype (n = 242) Low to moderate drinking (n = 62) Excessive drinking (n = 28) Males, n (%) 173 (71.5%) 47 (75.8%) 24 (85.7%) Age (years) 64.4 ± ± ± Smoking, n (%) 80 (33.1%) 25 (40.3%) 17 (60.7%)* Hypertension, n (%) 70 (28.9%) 17 (27.4%) 7 (25.0%) Diabetes, n (%) 45 (18.6%) 12 (19.4%) 7 (25.0%) TG (mmol/l) 1.80 ± ± ± HDL-C (mmol/l) 1.09 ± ± ± LDL-C (mmol/l) 2.10 ± ± ± ALT (U/L) 34.9 ± ± ± Creatinine (μmol/l) 84.3 ± ± ± Multivessel lesions, n (%) 77 (31.8%) 25 (40.3%) 11 (39.3%) Compared with group, *P < 0.05; compared with Low to moderate drinking group, P < 0.05). sus 92.2%). Furthermore, the mutated group (Table III) was divided into two subgroups (Table IV, ALDH2 1/ 2 and Table V, ALDH2 2/ 2). In the heterozygous mutated subgroup, smokers were more in excessive drinking group, while HDL-C level and percent of multivessel lesions were similar among no drinking and drinking groups (Table IV). The homozygous mutated subgroup

4 4 Xu, ET AL IntHeartJ Advance Publication Table IV. Effect of Alcohol Consumption on Coronary Artery Lesions in Patients with ALDH2 *1/*2 Mutated Genotype (n = 215) Low to moderate drinking (n = 53) Excessive drinking (n = 25) Males, n (%) 152 (70.7%) 39 (73.6%) 21 (84.0%) Age (years) 64.3 ± ± ± Smoking, n (%) 71 (33.0%) 21 (39.6%) 14 (56.0%)* Hypertension, n (%) 60 (27.9%) 15 (28.3%) 4 (16.0%) Diabetes, n (%) 36 (16.7%) 11 (20.8%) 7 (28.0%) TG (mmol/l) 1.81 ± ± ± HDL-C (mmol/l) 1.08 ± ± ± LDL-C (mmol/l) 2.10 ± ± ± ALT (U/L) 35.0 ± ± ± Creatinine (μmol/l) 85.4 ± ± ± Multivessel lesions, n (%) 65 (30.2%) 22 (41.5%) 9 (36.0%) Compared with group, *P < Table V. Effect of Alcohol Consumption on Coronary Artery Lesions in Patients with ALDH2 *2/*2 Mutated Genotype (n = 27) Drinking (n = 12) Males, n (%) 21 (77.8%) 11 (91.7%) Age (years) 65.6 ± ± Smoking, n (%) 9 (33.3%) 7 (58.3%) Hypertension, n (%) 10 (37.0%) 5 (41.7%) Diabetes, n (%) 9 (33.3%) 1 (8.3%) TG (mmol/l) 1.71 ± ± HDL-C (mmol/l) 1.16 ± ± LDL-C (mmol/l) 2.16 ± ± ALT (U/L) 33.9 ± ± 8.4* Creatinine (μmol/l) 75.9 ± ± Multivessel lesions, n (%) 12 (44.4%) 5 (41.7%) Compared with group, *P < consisting with 39 patients, and only 2 were excessive drinkers. Hence, we combined low to moderate drinking and excessive drinking into drinking group. In the homozygous group, HDL-C was no apparent increase among drinkers and non-drinkers, and there appear to exist substantial liver damages (reflected with ALT) among drinkers compared with non-drinkers, though ALT was also in the normal scale among drinkers (Table V). Coronary artery lesions also showed no difference between the two groups (Table V). Predictors of coronary multivessel lesions: Multivariate logistic regression analyses showed that male gender, aging, smoking, hypertension and ALDH2 mutation carriers were more susceptible to coronary multivessel lesions in this stable CAD cohort (Table VI). Furthermore, we divided the subjects into two groups by genotype, aging and smoking remained as risk factors while low to moderate drinking served as a protective factor and LDL-C as a risk factor for multivessel lesions in stable CAD patients with wild ALDH2 genotype, while none of these parameters but hypertension could predict multivessel lesions in stable CAD patients with mutated ALDH2 (Table VII). Low to moderate alcohol-induced HDL-C alterations in mice: The baseline HDL-C levels in the ALDH2 WT mice was higher than in the KO group (60.64 ± Table VI. Multivariate Logistic Regression Analyses for Predictors of Multivessel Lesions in All Subjects Variable Hazard Ratio (95% CI) Male 1.38 ( ) Elder age 1.02 ( ) Smoking 1.98 ( ) Hypertension 1.48 ( ) Diabetes 0.93 ( ) Low to moderate drinking 0.71 ( ) Excessive drinking 0.97 ( ) ALDH2 mutation 1.47 ( ) TG 1.02 ( ) HDL-C 0.91 ( ) LDL 1.19 ( ) mg/dl versus ± 6.87 mg/dl, P < 0.01, Figure). After alcohol consumption, HDL-C was increased in the WT mice (71.87 ± 9.53 mg/dl versus ± mg/dl, P < 0.01, Figure) but not in the KO mice (45.46 ± 7.67 mg/ dl versus ± 6.87 mg/dl, P > 0.05, Figure). Discussion The present study showed that stable CAD patients carrying mutated ALDH2 genotype were vulnerable to multiple coronary artery lesions. Fewer multivessel lesions and higher HDL-C levels were evidenced in ALDH2 wild genotype but not in ALDH2 mutated genotype. Multivariate logistic regression analyses showed that male gender, aging, and LDL-C level are risk factors, while low to moderate alcohol drinking was a protective factor for multivessel coronary artery lesion in ALDH2 wild genotype group, while these parameters could not predict multivessel coronary artery lesion but hypertension was a risk factor in ALDH2 mutated genotype group. Moreover, the animal experiment revealed that ALDH2 deficiency suppressed the positive effect of low to moderate alcohol on HDL-C level in WT mice. To the best of our knowledge, this is the first report showing the impact of ALDH2 genotype and alcohol consumption on coronary artery lesions in Chinese stable CAD patients. Previously, Krenz and Korthuis presented epidemi-

5 IntHeartJ Advance Publication ALDH2 AND ALCOHOL CONSUMPTION IN CHINESE CAD 5 Table VII. Multivariate Logistic Regression Analyses for Predictors of Multivessel Lesions in CAD Patients Wild and Mutated ALDH2 Genotypes ALDH2 *1/*1 ALDH2 *2 Variable Hazard Ratio Hazard Ratio (95% CI) (95% CI) Male 1.23 ( ) ( ) Elder age 1.03 ( ) ( ) Smoking 2.78 ( ) ( ) Hypertension 1.38 ( ) ( ) Diabetes 1.03 ( ) ( ) Low to moderate drinking 0.24 ( ) ( ) Excessive drinking 0.68 ( ) ( ) TG 0.87 ( ) ( ) HDL-C 0.98 ( ) ( ) LDL-C 1.41 ( ) ( ) Figure. HDL-C levels in four groups (WT-CON, ALDH2 wild type mice fed with water; WT-EOH, ALDH2 wild type mice fed with low to moderate ethanol; KO-CON, ALDH2 knockout mice fed with water; KO-EOH, ALDH2 knockout mice fed with low to moderate ethanol) ** P < NS indicates no significance. ologic evidence that moderate ethanol ingestion was linked to reduced cardiovascular morbidity. 20) However, Hines, et al. reported that alcohol intake may not protect against myocardial infarction patients with a defective ALDH2 genotype. 21) Recently, a survey involved Korean adults revealed that increased alcohol consumption was associated with an increased risk of coronary atherosclerosis for people with alcohol flushing phenomenon, which usually indicated subjects with ALDH2 mutated genotype. 22) A study with 410 Japanese coronary spasm patients found that ALDH2 2 variant could exacerbate coronary spasm. 23) Consistent with these findings, our study indicated that the mutated ALDH2 carriers were more likely to suffer with multivessel lesions irrelevant to alcohol consumption. Thus, the protection of low to moderate alcohol consumption for CAD may be disappeared in the context of ALDH2 mutated genotype. Association between alcohol consumption and an increase in HDL level has been well documented. 24,25) As early as in 1981, Gordon, et al. reported that the HDL-C level was affected by alcohol consumption. 26) Previous studies have suggested that HDL-C is a protective factor for CAD, which may represent the main mechanism of alcohol-induced cardiac protection. 26,27) However, the positive role of alcohol consumption on HDL-C was inhibited in ALDH2 KO mice in our study. Similarly, in CAD patients with ALDH2 mutation, low to moderate alcohol consumption had little influence on HDL-C, while HDL-C level was significantly higher in low to moderate alcohol consumption group than in no drinking group for CAD patients with wild type ALDH2. In the case of ALDH2 mutation, alcohol consumption could not play a protective role in the cardiovascular system maybe because the beneficial effect of ethanol intake, especially the low to moderate alcohol consumption, on HDL-C was depressed. Previous studies found HDL-C was related to the ALDH2 genotype, and ALDH2 genetic variation can influence HDL-C level. 28) ALDH2 homozygous genotype was associated with lower HDL-C concentrations compared with wild genotype. 29) In our animal experiment, HDL-C in ALDH2 knockout mice was lower than in wild type mice at baseline. However, in non-drinkers of the human study, HDL-C in ALDH2 homozygous genotype was a little higher than the wild genotype, which may be explained by the obvious difference in sample size. Recent studies have proved that ALDH2 mutated genotype is a risk factor for CAD through multiple mechanisms. 14,30) And the ALDH2 mutation has been shown to be associated with certain traditional cardiovascular risk factors, such as dyslipidemia, hypertension, and diabetes mellitus or hyperglycemia. 7,15) Hence, generally recognized risk factors for CAD may not be applicable in ALDH2 mutated population. Interestingly, some data suggest that the ALDH2 mutation may be a protective factor for hypertension. 30) In other words, higher incidence of hypertension is not covariant with ALDH2 mutation, which may be part of the reason that hypertension instead served as a strong predictor for severity of CAD in ALDH2 mutated population. In summary, ALDH2 deficiency aggravated coronary artery atherosclerosis in CAD patients, partly due to the fact that ALDH2 deficiency might suppress the positive effect of low to moderate alcohol on HDL-C level. Limitation: Inaccuracy in the self-reporting of drinking may potentially limit the value of the present study, since such a system may underestimate or overestimate the true

6 6 Xu, ET AL IntHeartJ Advance Publication alcohol intake status, especially in men who actually consume large amounts of alcohol. Although participants in this study were enrolled consecutively, it cannot escape from the arguments of selection bias and/or substantial influences by confounding factors. And the study subjects consisted of mainly male CAD patients, and most of the ALDH2 mutation carriers were heterozygote, which may narrow the phenotype gap between the wild type and the homozygote. In addition, HDL-C levels were confounded by smoking which was correlated with the degree of alcohol consumption. Therefore, future studies with population-based cohort studies including larger patient samples both for CAD patients and subjects carrying higher risk for CAD are warranted to define the role of ALDH2 genotype and alcohol consumption as well as their combined impact on HDL-C and CAD progression. 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