Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

Akira Yokoyama; Tetsuji Yokoyama; Mitsuru Kimura; Sachio Matsushita; Masako Yokoyama

doi:10.1371/journal.pone.0255276

. 2021 Jul 26;16(7):e0255276. doi: 10.1371/journal.pone.0255276

Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

Akira Yokoyama ^1,^*, Tetsuji Yokoyama ², Mitsuru Kimura ¹, Sachio Matsushita ¹, Masako Yokoyama ³

Editor: Nadine Bernhardt⁴

PMCID: PMC8312924 PMID: 34310648

Abstract

Objective

The risk of alcohol dependence (AD) in Japanese men and women was evaluated according to combinations of alcohol flushing and aldehyde dehydrogenase-2 (ALDH2, rs671) and alcohol dehydrogenase-1B (ADH1B, rs1229984) genotypes, all of which are known to determine AD susceptibility in Asians. Previous studies have focused on men, since women account for a smaller proportion of AD subjects.

Methods

Case control studies were conducted between 3721 male and 335 female AD Japanese and 610 male and 406 female controls who were asked about their current or former tendency to experience facial flushing after drinking a glass of beer and underwent ALDH2 and ADH1B genotyping. The time at which alcohol-induced facial flushing tendencies had disappeared in former-flushing AD subjects was also evaluated.

Results

Current alcohol flushing, the inactive ALDH2*1/*2 genotype, and the fast-metabolizing ADH1B*2 allele were less frequently found in the AD groups. Although alcohol flushing was strongly influenced by the ALDH2 and ADH1B genotypes, multiple logistic model showed that never or former flushing and the genotype combinations were independent strong risk factors of AD in men and women. Never or former flushing (vs. current flushing) markedly increased the odds ratios of AD in carriers of each of the ALDH2 and ADH1B genotype combinations. The temporal profiles for drinking and flushing in former-flushing AD subjects revealed that the flushing response disappeared soon after or before the start of habitual drinking during young adulthood, regardless of the ALDH2 genotype.

Conclusion

Although alcohol flushing is influenced by the ALDH2 and ADH1B genotypes, constitutional or acquired flushing tolerance is an independent susceptibility trait for AD. The combination of the alcohol flushing status and the ALDH2 and ADH1B genotypes can provide a better new strategy for AD risk assessment than the alcohol flushing status alone or the genotypes alone in Asian men and women.

Introduction

Alcohol dehydrogenases (ADHs) oxidize ethanol to acetaldehyde, and then aldehyde dehydrogenases (ALDHs) oxidize acetaldehyde to acetate. The inactive ALDH2*2 allele (rs671), which results in slow acetaldehyde elimination, and the fast-metabolizing ADH1B*2 allele (rs1229984), which results in fast acetaldehyde production, both enhance alcohol-induced facial flushing [1–6] and are strong Asian protective factors against alcohol dependence [7–10]. ALDH2 plus ADH1B genotyping using buccal smear DNA is commercially available and inexpensive in Japan. Knowing an individual’s own ALDH2 plus ADH1B genotype is a growing preventive strategy against alcohol dependence.

However, alcohol-dependent individuals generally experience a weak or absent alcohol-induced facial flushing tendency, regardless of their ALDH2 and ADH1B genotypes; for example, 86.4% of Japanese alcohol-dependent men with the inactive heterozygous ALDH2*1/*2 genotype did not report current flushing and instead reported never or former flushing [6]. We empirically noticed that patients with alcohol dependence who were never or former flushers were often surprised to learn that they had inactive ALDH2, because many never flushers had believed that they had active ALDH2 and many former flushers had experienced alcohol flushing for only short periods of time during their young adulthood [6]. The combination of the alcohol flushing status and ALDH2 plus ADH1B genotype may have a better performance for predicting the alcohol-dependence risk in comparison with alcohol flushing alone or genotyping alone.

The time at which an alcohol-induced facial flushing tendency disappeared in former-flushing, alcohol-dependent subjects should also be evaluated. Most of the research in this field has focused on alcohol-dependent men, since women accounted for a very small proportion of alcohol-dependent patients among Asians [11].

According to a report, alcohol consumption in inactive ALDH2*1/*2 carriers in the general population increased in the order of current flushers, former flushers, and never flushers [12]. If the flushing disappearance occurred in young adulthood and not around the time of development of alcohol dependence in many former flushers who are alcohol-dependent, it is conceivable that both former flushing and never flushing may serve as independent risk factors for alcohol dependence, in combination with the ADH1B and ALDH2 genotypes.

To evaluate these issues, we compared a large dataset of Japanese alcohol-dependent men and women with historical control groups of Japanese men and women who had undergone annual health checkups. In addition, we evaluated the temporal profiles of drinking and flushing in former-flushers with alcohol dependence.

Materials and methods

Subjects

The study subjects were 3721 Japanese men and 335 Japanese women aged 40–79 years with alcohol dependence who met the following criteria: (a) visited the Kurihama Medical and Addiction Center for treatment of alcohol dependence for the first time between 2004 and 2017 in men and between 2006 and 2017 in women, (b) were asked about their alcohol flushing, and (c) underwent routine ADH1B and ALDH2 genotyping. The questions were asked with both the questioners and the patients having no knowledge of the patients’ ADH1B and ALDH2 genotypes. All of the subjects met the ICD-10 or DSM-IV criteria for alcohol dependence [13, 14]. Male subjects with alcohol dependence were a subgroup (40–79 years) of 4051 subjects aged 30–79 years, in which we recently reported the associations between the results of an alcohol flushing questionnaire and the ALDH1B and ALDH2 genotypes [6]. We borrowed historical control data of 1016 Japanese subjects (610 men and 406 women) aged 40–79 years who had visited two Tokyo clinics for annual health checkups and had registered in case control studies of esophageal cancer as cancer-free controls between 2000 and 2001 [12, 15]. Most of the controls were ordinary residents or workers lived in Tokyo or neighboring areas. Kanagawa, where our center is located, is situated next to Tokyo, and previous studies have shown similar ADH1B and ALDH2 genotype distributions in Kanagawa and Tokyo [12, 15]. Although alcohol-dependent individuals had not been excluded from the controls, the life-time frequency of alcohol dependence based on ICD-10 criteria has been estimated to be only 1.5% in men and 0.2% in women among Japanese adults according to 2003 nationwide survey [11]. The anonymous historical control data consisted of age, results of the flushing questionnaire, and the ADH1B and ALDH2 genotypes.

Between July 2019 and December 2020, 283 Japanese subjects with alcohol dependence (253 men and 30 women) aged 40–79 years who visited the Center for the first time were asked about their alcohol flushing history and underwent routine ADH1B and ALDH2 genotyping. Of the subjects, 7 (6 men and one women), 69 (64 men and 5 women), and 207 (183 men and 24 women) reported themselves as current flushers, former flushers, and never flushers, respectively. We asked 66 former flushers (61 men and 5 women) the following question: “Up until what age did you have a tendency to develop facial flushing immediately after drinking a glass of beer?”

The ethics committee of the Kurihama Medical and Addiction Center reviewed and approved the proposed study, and all procedures involved in this study were performed in accordance with the Declaration of Helsinki with written informed consent from each participant.

The simple flushing questionnaire

The physicians verbally questioned the alcohol-dependent subjects about flushing using two simple questions [12]: (a) “Do you have a tendency to flush in the face immediately after drinking a glass of beer?” and (b) “Did you have a tendency to flush in the face immediately after drinking a glass of beer during the first 1–2 years after you started drinking?” A “glass” means about 180 ml, the volume of the most common size Japanese beer glass. The term “current flushing” was applied to individuals who answered “yes” to question (a), and the term “former flushing” to those who answered “no” to question (a) and “yes” to question (b). The term “never flushing” was applied to the remaining subjects. The control subjects had completed a self-reported questionnaire consisting of the same questions.

ALDH2 and ADH1B genotypes

The ALDH2 and ADH1B genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism methods on blood DNA samples obtained from the subjects [16, 17].

Statistical analyses

Data were summarized as the mean and standard deviation (SD) or the percentage values and compared between alcohol dependence and control groups by Student’s t-test or χ² test, respectively. Age-adjusted percentage values were calculated by the direct method and statistically tested by the Cochran-Mantel-Haenszel test. Multivariate odds ratios (ORs) and the 95% confidence intervals (CIs) were estimated using multiple logistic models. A p value of <0.05 was considered as statistically significant. All the statistical analyses were performed using the SAS statistical analysis software (version 9.4; SAS Institute, Cary, NC).

Results

Table 1 shows the age distribution, the results of the simple flushing questionnaire, and the ALDH2 and ADH1B genotype combinations in men and women, respectively. Since significantly different age distribution was observed between the alcohol-dependent groups and the controls, the subsequent multivariate analyses were performed with age-adjustment. Striking differences in the results of the flushing questionnaire and the ALDH2 and ADH1B genotypes were shown between the alcohol-dependent groups and the controls regardless of gender. The inactive ALDH2*2/*2 homozygotes were not found in the alcohol-dependent groups, while in 6.9% and 5.4% of the male and female controls, respectively. Current alcohol flushing, the inactive ALDH2*1/*2 genotype, and the fast-metabolizing ADH1B*2 allele were less frequently found similarly in the alcohol-dependent men and women groups.

Table 1. Alcohol flushing and ADH1B and ALDH2 genotypes in the male and female subjects.

		Men				P	Women				P
		Alcohol dependence (n = 3721)		Controls (n = 610)			Alcohol dependence (n = 335)		Controls (n = 406)
		n	%	N	%		n	%	n	%
Age
40–49		1077	28.9%	41	6.7%	<0.0001^a	155	46.3%	29	7.1%	<0.0001 ^a
50–59		1266	34.0%	285	46.7%		110	32.8%	202	49.8%
60–69		983	26.4%	254	41.6%		56	16.7%	144	35.5%
70–79		395	10.6%	30	4.9%		14	4.2%	31	7.6%
Mean±SD		56.2±9.7		58.9±7.1		<0.0001^b	52.2±9.0		58.7±7.6		<0.0001^b
Alcohol flushing
Never flushing		3037	81.6%	315	51.6%^*	<0.0001^c	296	88.4%	226	55.7%^*	<0.0001^c
Former flushing		553	14.9%	68	11.1%^*		31	9.3%	26	6.4%
Current flushing		131	3.5%	227	37.2%^*		8	2.4%	154	37.9%^*
ALDH2 genotype	ADH1B genotype
1/1 (active)	1/1 (slow)	845	22.7%	11	1.8%^*	<0.0001^c	87	26.0%	13	3.2%^*	<0.0001^c
	1/2 (fast)	1061	28.5%	121	19.8%^*		104	31.0%	75	18.5%^*
	2/2 (fast)	1215	32.7%	194	31.8%		102	30.4%	133	32.8%
1/2 (inactive)	1/1 (slow)	194	5.2%	17	2.8%^*		18	5.4%	13	3.2%
	1/2 (fast)	181	4.9%	78	12.8%^*		8	2.4%	63	15.5%^*
	2/2 (fast)	225	6.0%	147	24.1%^*		16	4.8%	87	21.4%^*
2/2 (inactive)	Any	0	0.0%	42	6.9%^*		0	0.0%	22	5.4%^*

Open in a new tab

ADH1B, alcohol dehydrogenase-1B; ALDH2, aldehyde dehydrogenase-2

^a χ² test;

^b Student’s t-test;

^c Cochran-Mantel-Haenszel test adjusted for age.

* P <0.05 for comparing the prevalence of each category between alcohol dependence and controls. ^c

Figs 1 and 2 show the comparison of the alcohol flushing according to the combination of ALDH2 and ADH1B genotypes in men and women, respectively. In comparison with the controls, never or former flushing was more frequently reported in the alcohol-dependent men with any combinations of the ADH1B and ALDH2 genotypes, and in the alcohol-dependent women with the ALDH2*1/*2 genotype.

Fig 1 — Never or former flushing was more frequently reported in the alcohol-dependent men than in the controls regardless of the *ALDH2* and *ADH1B* genotype combination. Percentage values were adjusted for age by a direct method using the alcohol dependence group as the standard population; p values were by Cochran-Mantel-Haenszel test adjusted for age.

Fig 2 — Never or former flushing was more frequently reported in the alcohol-dependent women than in the controls among the *ALDH2*1/*2* genotype carrier. Percentage values were adjusted for age by a direct method using the alcohol dependence group as the standard population; p values were by Cochran-Mantel-Haenszel test adjusted for age.

Tables 2 and 3 show the age-adjusted ORs and 95% CIs of alcohol dependence according to the reported alcohol flushing status and combinations of the ALDH2 and ADH1B genotypes using multiple logistic regression models in men and women, respectively. When current flushing was used as a reference, never flushing (OR [95% CI] = 18.1 [13.9–23.5] in men and 21.2 [9.89–45.6] in women) and former flushing (15.5 [10.9–22.0] in men and 19.2 [7.52–49.0] in women) markedly increased the risk of alcohol dependence. When the ALDH2*1/*2 and ADH1B*2/*2 combination was used as a reference, the increased number of ALDH2*1 allele and ADH1B*1 allele in the genotype combinations resulted in the higher ORs of alcohol dependence, and the OR (95%CI) by the ADH1B*1/*1 and ALDH2*1/*1 combination was the highest (46.7 [24.8–88.2] in men and 31.8 [13.7–73.8] in women). A multivariate model including both the alcohol flushing status and the ALDH2 and ADH1B genotype combinations showed that the alcohol flushing status and the ADH1B and ALDH2 genotype combinations were strong independent strong risk factors of alcohol dependence in both men and women, though each OR in this model was lower than that in a model that included the alcohol flushing status alone or the genotype combinations alone.

Table 2. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to alcohol flushing status and/or the combinations of ALDH2 and ADH1B genotypes in the male subjects.

		Age-adjusted OR (95%CI) of alcohol dependence
		Number of AD/control	Flushing alone	Genotype alone	Flushing and Genotype
Alcohol flushing
Never flushing		3037/315	18.1 (13.9–23.5)	-	7.30 (5.13–10.4)
Former flushing		553/68	15.5 (10.9–22.0)	-	10.6 (7.37–15.3)
Current flushing		131/227	1 (ref.)	-	1 (ref.)
ALDH2 genotype	ADH1B genotype
1/1 (active)	1/1 (slow)	845/11	-	46.7 (24.8–88.2)	23.6 (11.9–46.8)
	1/2 (fast)	1061/121	-	5.64 (4.22–7.53)	2.88 (1.97–4.21)
	2/2 (fast)	1215/194	-	4.00 (3.07–5.23)	2.05 (1.42–2.95)
1/2 (inactive)	1/1 (slow)	194/17	-	7.24 (4.19–12.5)	4.34 (2.41–7.82)
	1/2 (fast)	181/78	-	1.42 (1.00–2.01)	1.27 (0.85–1.90)
	2/2 (fast)	225/147	-	1 (ref.)	1 (ref.)
2/2 (inactive)	Any	0/42	-	0 (NA)	0 (NA)

Open in a new tab

ADH1B, alcohol dehydrogenase-1B; ALDH2, aldehyde dehydrogenase-2; NA, not applicable

Odds ratio (OR) and 95% confidence interval (CI) were estimated by a logistic regression model.

- Not included in the model.

Table 3. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to alcohol flushing status and/or the combinations of ALDH2 and ADH1B genotypes in the female subjects.

		Age-adjusted OR (95%CI) of alcohol dependence
		Number of AD/control	Flushing alone	Genotype alone	Flushing and genotype
Alcohol flushing
Never flushing		296/226	21.2 (9.89–45.6)	-	7.57 (3.04–18.8)
Former flushing		31/26	19.2 (7.52–49.0)	-	14.0 (5.26–37.5)
Current flushing		8/154	1 (ref.)	-	1 (ref.)
ALDH2 genotype	ADH1B genotype
1/1 (active)	1/1 (slow)	87/13	-	31.8 (13.7–73.8)	15.8 (5.84–42.8)
	1/2 (fast)	104/75	-	6.71 (3.44–13.1)	3.24 (1.40–7.51)
	2/2 (fast)	102/133	-	3.66 (1.91–7.00)	1.78 (0.78–4.04)
1/2 (inactive)	1/1 (slow)	18/13	-	6.79 (2.55–18.1)	4.85 (1.62–14.6)
	1/2 (fast)	8/63	-	0.56 (0.21–1.51)	0.56 (0.18–1.73)
	2/2 (fast)	16/87	-	1 (ref.)	1 (ref.)
2/2 (inactive)	Any	0/22	-	0 (NA)	0 (NA)

Open in a new tab

AD, alcohol dependence; ADH1B, alcohol dehydrogenase-1B; ALDH2, aldehyde dehydrogenase-2; NA, not applicable

Odds ratio (OR) and 95% confidence interval (CI) were estimated by a logistic regression model.

- Not included in the model.

Table 4 shows the age-adjusted ORs of alcohol dependence according to the combinations of reported alcohol flushing and ALDH2 and ADH1B genotype combinations in men. When the combination of the current flushing, ALDH2*1/*2, and ADH1B*2/*2 was used as a reference, the never or former flushing was markedly increased the ORs of alcohol dependence in the carriers of each ALDH2 and ADH1B genotype combination. Although the estimated ORs were unstable due to the small number in current flushing category in alcohol-dependent women with each genotype combination, similar results were obtained in women (Table 5).

Table 4. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to the combinations of alcohol flushing status and ALDH2 and ADH1B genotypes in the male subjects.

ALDH2 genotype	ADH1B genotype	Alcohol flushing
		Current flushing		Former flushing		Never flushing
		Number of AD/control	OR (95%CI)	Number of AD/control	OR (95%CI)	Number of AD/control	OR (95%CI)
1/1 (active)	1/1 (slow)	8/1	17.0 (1.92–150)	47/1	138 (18.2–999<)	790/9	241 (112–517)
	1/2 (fast)	23/4	12.9 (4.04–41.5)	89/3	78.0 (23.0–265)	949/114	24.3 (15.7–37.4)
	2/2 (fast)	19/11	5.88 (2.50–13.9)	93/19	13.0 (6.86–24.6)	1103/164	19.2 (12.6–29.3)
1/2 (inactive)	1/1 (slow)	14/5	7.40 (2.38–23.1)	79/6	42.3 (16.8–106)	101/6	43.1 (17.2–108)
	1/2 (fast)	25/57	1.12 (0.59–2.10)	116/12	27.5 (13.4–56.2)	40/9	11.2 (4.82–26.0)
	2/2 (fast)	42/108	1 (ref.)	129/27	14.7 (8.29–26.2)	54/12	13.7 (6.45–28.9)

Open in a new tab

AD, alcohol dependence; ADH1B, alcohol dehydrogenase-1B; ALDH2, aldehyde dehydrogenase-2; NA, not applicable

Age-adjusted odds ratio (OR) and 95% confidence interval (CI) were estimated by a logistic regression model using ALDH2*1/*2 (inactive) plus ADH1B*2/*2 (fast) plus current flushing as the reference group.

Table 5. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to the combinations of alcohol flushing status and ALDH2 and ADH1B genotypes in the female subjects.

ALDH2 genotype	ADH1B genotype	Alcohol flushing
		Current flushing		Former flushing		Never flushing
		Number of AD/control	OR (95%CI)	Number of AD/control	OR (95%CI)	Number of AD/control	OR (95%CI)
1/1 (active)	1/1 (slow)	1/0	NA	0/0	-	86/13	346 (42.8–999<)
	1/2 (fast)	2/2	54.8 (2.89–999)	7/3	111 (9.19–999<)	95/70	73.9 (9.74–561)
	2/2 (fast)	2/7	18.2 (1.32–251)	6/6	68.3 (6.61–704)	94/120	40.6 (5.38–306)
1/2 (inactive)	1/1 (slow)	1/7	7.47 (0.36–153)	4/2	111 (7.30–999<)	13/4	177 (17.3–999<)
	1/2 (fast)	1/49	1.14 (0.07–19.4)	5/6	33.2 (2.94–374)	2/8	11.6 (0.82–163)
	2/2 (fast)	1/68	1 (ref.)	9/9	52.6 (5.57–497)	6/10	34.6 (3.51–342)

Open in a new tab

AD, alcohol dependence; ADH1B, alcohol dehydrogenase-1B; ALDH2, aldehyde dehydrogenase-2; NA, not applicable

Table 6 shows the temporal profiles for drinking and flushing in 66 former-flushing, alcohol-dependent subjects according to their ALDH2 genotypes. No differences in age, age at the start of drinking, age up until which the flushing tendency continued, duration of flushing, age at the start of habitual drinking, and temporal profiles between alcohol flushing and habitual drinking were seen according to ALDH2 genotype. The mean age up until which the flushing tendency continued, the mean age at the start of habitual drinking, and the mean age at the first hospital visit for alcohol-related problems were 25.7±6.1 years, 25.9±7.4 years, and 51.4±12.4 years, respectively. The flushing tendency had disappeared before and within 5 years of the start of habitual drinking in 30 (45.5%) and 24 (36.4%) of the 66 former-flushing subjects, respectively.

Table 6. Temporal profiles for drinking and flushing in former-flushing, alcohol-dependent subjects according to ALDH2 genotype.

	Total	ALDH2 genotype
	Total	1/1 (active)	1/2 + 2/2 (inactive)	P
Number of patients (61 men and 5 women)	66	28	38
Age (years)	53.9±10.9	53.4±11.4	54.3±10.5	0.72
Age at the first hospital visit for alcohol-related problems^a	51.4±12.4	50.9±13.6	51.8±11.5	0.77
Age at the start of drinking (years)	18.8±4.3	18.9±3.9	18.7±4.7	0.87
Age up until which the flushing tendency continued (years)	25.7±6.1	24.6±7.2	26.5±5.1	0.24
Duration of flushing tendency (years)	8.0±5.9	6.8±6.5	8.8±5.4	0.16
Age at the start of habitual drinking (years)	25.9±7.4	25.4±8.3	26.2±6.7	0.23
Disappearance of flushing tendency
Before the start of habitual drinking	45.5%	42.9%	47.4%	0.91
Within 5 years after the start of habitual drinking	36.4%	39.3%	34.2%
More than 6 years after the start of habitual drinking	18.2%	17.9%	18.4%

Open in a new tab

ALDH2, aldehyde dehydrogenase-2. Data are expressed as the mean ± SD and percentage values. N = 1 for ALDH2*2/*2.

^a Alcohol-related organ injuries or alcohol dependence.

Discussion

The present study showed that the never or former flushing by the simple flushing questionnaire was a strong independent risk factor of alcohol dependence in any ALDH2 and ADH1B genotype combination carriers in both men and women. The increased number of ALDH2*1 allele and ADH1B*1 allele in the ALDH2 and ADH1B genotype combinations resulted in the higher risk of alcohol dependence in women as well as men, and similar ORs of alcohol dependence was observed between women and men. Flushing responses after a small amount of drinking have been mainly attributable to high acetaldehyde exposure, and are influenced by the ALDH2 and ADH1B genotypes [1–6]. Current flushers were less frequently reported in the subject groups with alcohol dependence than in the control group, and never flushers and former flushers showed similarly elevated ORs for alcohol dependence as compared to current flushers, among both men and women. However, the flushing status subdivided subjects with each of the ALDH2 and ADH1B genotypes into groups with substantially lower and higher ORs. Never or former flushing markedly increased the ORs of alcohol dependence among carriers of any ALDH2 and ADH1B genotype combination, compared with current flushing. Commercially available ALDH2 plus ADH1B genotyping has been used as a preventive tool against alcohol dependence in Japan. Combined evaluation of the alcohol flushing status and the ALDH2 plus ADH1B genotype may show superior ability than that of either alone for predicting the risk of alcohol dependence.

The temporal profiles of drinking and flushing were evaluated, for the first time, in former flushers with alcohol dependence; these profiles revealed that the flushing tendency disappeared soon after or before the start of habitual drinking, regardless of the ALDH2 genotype. These results suggest that constitutional or acquired tolerance against alcohol flushing after a small amount of drinking is another independent susceptibility trait for alcohol dependence. Tolerance against alcohol flushing may be acquired due to a pattern of heavy alcohol use in some subjects during their young adulthood, and disappearance of flushing in some former flushers could have been a consequence of alcohol dependence rather than a risk factor for alcohol dependence. It would be more relevant to study detailed drinking behavior during the young adulthood. However, the flushing tendency disappeared before the start of habitual drinking in 45.5% of former flushers, and this interpretation was not applicable to never flushers among inactive ALDH2*1/*2 carriers. The mean age until which the flushing tendency continued and the mean age at the first hospital visit for alcohol-related problems were 25.7 years and 51.4 years, respectively. Thus, use of alcohol flushing data in addition to ALDH2 genotyping is useful to predict, and could be expected to reduce, the risk of future development of alcohol dependence, especially in younger generations. Combined use of this information with established screening tools, e.g., Alcohol Use Disorders Identification Test (AUDIT; [18]), may be more useful to detect alcohol use disorders that are already present.

It was also puzzling that there were no differences in the temporal profiles of the disappearance of alcohol flushing according to the ALDH2 genotypes. These results suggest the presence of other unidentified factors that prevent the alcohol flushing tendency. A cutaneous flushing, increase in skin temperature, decrease in blood pressure after drinking have been reported to be significantly blocked by the administration of antihistamine H1 and H2 receptor antagonists in Asian flushers [19–22]. Aspirin [23] and opioid antagonist, nalmefene [24], also weaken some flushing responses in Asian flushers. As well as the pathways via the central and peripheral histamine, prostaglandins, and opioid receptors, several cascades from the triggers of ethanol and acetaldehyde to the final flushing responses may participate in the flushing responses. Further evaluation of the background of the never or former flushing trait is warranted in future research, combining flushing-related genomics and drinking pattern in young adulthood.

Inactive ALDH2 has been reported as a strong risk factor for squamous cell carcinoma of the upper aerodigestive tract in East Asian drinkers [25, 26]. Current or former flushing has been shown to be a predictor of the risk of cancer as a surrogate marker of inactive ALDH2 in various drinking populations [27]. However, the cancer risk in the current or former flushers was substantially lower than the risk in ALDH2*1/*2 carriers in an alcohol-dependent population overall [27]. This could be partly attributable to the existence of additional causes of alcohol flushing besides the ALDH2 genotype in persons susceptible to alcohol dependence. Investigation is warranted, in a future study, of whether and how the combination of alcohol flushing status and the ALDH2 plus ADH1B genotype might affect drinking behaviors and manifestations of alcohol-related problems.

The present study had several limitations. This case control study was based on the results of questions about facial flushing tendency after a glass of beer. Recall bias of the responses to the questionnaire cannot be ruled out, and the results regarding associations of alcohol flushing with ALDH2 genotype and susceptibility to alcohol dependence differ according to how the question about alcohol flushing is posed. When Japanese subjects were asked about current facial flushing after drinking alcohol without specifying the alcohol dose, half of those with active ALDH2 were sometimes or always flushers, because they experienced facial flushing after drinking a substantial amount of alcohol [28], and the sometimes flushers showed the highest risk for alcohol abuse [29]. However, when current or former flushing individuals after a glass of beer in the present questionnaire were assumed to have the inactive ALDH2*2 allele, alcohol flushing was found to serve as a surrogate marker of the ALDH2*2 allele carriers with a sensitivity and specificity of approximately 90% in the Japanese general population [12, 15, 30]. In Japan, it is customary to toast each other with a glass (≈180 mL) of beer at social gatherings. Cultural differences, including popular alcoholic beverage types and typical sizes of beer glasses, could influence the results of the flushing questionnaire. The sensitivity and specificity of the questionnaire have been previously reported as 79%-95% and 77%-82%, respectively, in Korea [31] and 89% and 81%, respectively, in Taiwan [32]. Caucasians who reported flushing responses after 1 or 2 drink, which is equivalent to approximately 2 or 3 typical Japanese glasses of beer, were more likely to have symptoms of alcohol dependence, and report a parental history of alcohol problems [33]. Such Caucasians’ flushing after moderate amounts of alcohol may be a risk factor in contrast with the protective role of the Japanese flushing after a small amount of alcohol. An oral alcohol challenge test using 0.56 g ethanol/kg showed lower specificity of self-reported flushing for predicting the presence of inactive ALDH2 in comparison to investigator-observed flushing [34]. Self-reported flushing may be a net self-evaluation of the perception of general discomfort [35] and cardiovascular effects [36] in flushing responses.

Another limitation was that alcohol dependence had not been excluded from the historical controls who had registered in case control studies of esophageal cancer as cancer-free controls between 2000 and 2001 [12, 15, 37]. The proportion of control subjects who drank alcohol on 3 or more days per week and consumed at least 22 g of ethanol at a time was 56.0% in men [37] and 12.9% in women [15]. These values were similar to the 53.3% in male adults and 9.1% in female adults reported by the National Nutrition Survey in 2001 [38]. A 2003 nationwide survey showed a low prevalence (1.5% in men and 0.2% in women) of lifetime alcohol dependence diagnosed according to the ICD-10 criteria in Japanese adults [11]. The mortality rate associated with esophageal cancer per 100,000 in 2001 was 10.5 in men and 1.3 in women [39]. Thus, although the ADH1B and ALDH2 genotypes are risk factors for both alcohol dependence [6–10] and esophageal cancer [15, 25, 37], it is unlikely that the potential selection bias of the controls, i.e., esophageal-cancer-free and imperfect exclusion of alcohol dependence, significantly affected the present results due to their low prevalence.

Although the total number of alcohol-dependent patients was large, the numbers in some categories of combinations of alcohol flushing status and genotypes were small, especially for the female patients and the control groups. In addition to the retrospective self-reported alcohol flushing questionnaire, the temporal pattern of former flushing also has potential recall bias. Furthermore, it was evaluated mainly in male subjects with alcohol dependence, which may differ from the case in the general population and women. There is a higher probability of Chinese university students thinking that they should encourage a flusher to stop drinking or drink less when the flusher is a woman [40], which may prevent the development of tolerance in female flushers. Generalization of the results obtained in this study, which was based on investigations of treatment-seeking alcohol-dependent patients, will require confirmation in various heavy-drinking populations, including groups with mild alcohol-dependent cases and a large number female cases, as well as in large control populations.

In conclusion, never flushing and former flushing were identified as strong independent risk factors for alcohol dependence in carriers of any ALDH2 and ADH1B genotypes, and combined evaluation of the self-reported alcohol flushing history and the ALDH2 and ADH1B genotypes might enable better estimation of the risk of alcohol dependence in Asians.

Supporting information

S1 Data

(XLSX)

Click here for additional data file.^{(142.7KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The Authors received no specific funding for this work.

References

1.Takeshita T, Mao XQ, Morimoto K. The contribution of polymorphism in the alcohol dehydrogenase β subunit to alcohol sensitivity in a Japanese population. Hum Genet 1996; 97: 409–13. doi: 10.1007/BF02267057 [DOI] [PubMed] [Google Scholar]
2.Chen WJ, Chen CC, Yu JM, Cheng AT. Self-reported flushing and genotypes of ALDH2, ADH2, and ADH3 among Taiwanese Han. Alcohol Clin Exp Res 1998; 22: 1048–52. [PubMed] [Google Scholar]
3.Matsuo K, Wakai K, Hirose K, Saito T, Tajima K. Alcohol dehydrogenase 2 His47Arg polymorphism influences drinking habit independently of aldehyde dehydrogenase 2 Glu487Lys polymorphism: analysis of 2,299 Japanese subjects. Cancer Epidemiol Biomarkers Prev 2006; 15: 1009–13. doi: 10.1158/1055-9965.EPI-05-0911 [DOI] [PubMed] [Google Scholar]
4.Luczak SE, Pandika D, Shea SH, Eng MY, Liang T, Wall TL. ALDH2 and ADH1B interactions in retrospective reports of low-dose reactions and initial sensitivity to alcohol in Asian American college students. Alcohol Clin Exp Res 2011; 35: 1238–45. doi: 10.1111/j.1530-0277.2011.01458.x [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Hayashida M, Kamada Y, Ota T, Kojima S, Iwao-Koizumi K, Murata S, et al. Associations between ALDH2 and ADH1B Genotypes and Ethanol-Induced Cutaneous Erythema in Young Japanese Women. Nihon Eiseigaku Zasshi 2015; 70: 134–8 (in Japanese with English abstract). [DOI] [PubMed] [Google Scholar]
6.Yokoyama A, Yokoyama T, Matsui T, Mizukami T, Kimura M, Matsushita S, et al. Impacts of interactions between ADH1B and ALDH2 genotypes on alcohol flushing, alcohol reeking on the day after drinking, and age distribution in Japanese alcohol-dependent men. Pharmacogenet Genomics 2020; 30: 54–60. doi: 10.1097/FPC.0000000000000395 [DOI] [PubMed] [Google Scholar]
7.Higuchi S, Matsushita S, Muramatsu T, Kinoshita T, Takagi S, Kono H. Alcohol and aldehyde dehydrogenase polymorphism and the risk for alcoholism. Am J Psychiatry 1995. 152, 1219–1221. doi: 10.1176/ajp.152.8.1219 [DOI] [PubMed] [Google Scholar]
8.Chen CC, Lu RB, Chen YC, Wang MF, Chang YC, Li TK, et al. Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism. Am J Hum Genet 1999; 65: 795–807. doi: 10.1086/302540 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Luczak SE, Glatt SJ, Wall TL. Meta-analyses of ALDH2 and ADH1B with alcohol dependence in Asians. Psychol Bull 2006; 132: 607–21. doi: 10.1037/0033-2909.132.4.607 [DOI] [PubMed] [Google Scholar]
10.Kim DJ, Choi IG, Park BL, Lee BC, Ham BJ, Yoon S, et al. Major genetic components underlying alcoholism in Korean population. Hum Mole Genetics 2008; 17: 854–8. doi: 10.1093/hmg/ddm357 [DOI] [PubMed] [Google Scholar]
11.Osaki Y, Kinjo A, Higuchi S, Matsumoto H, Yuzuriha T, Horie Y, et al. Prevalence and trends in alcohol dependence and alcohol use disorders in Japanese adults; Results from periodical nationwide surveys. Alcohol Alcohol 2016; 51: 465–73. doi: 10.1093/alcalc/agw002 [DOI] [PubMed] [Google Scholar]
12.Yokoyama T, Yokoyama A, Kato H, Tsujinaka T, Muto M, Omori T, et al. Alcohol flushing, alcohol and aldehyde dehydrogenase genotypes, and risk for esophageal squamous cell carcinoma in Japanese men. Cancer Epidemiol Biomarkers Prev 2003; 12: 1227–33. [PubMed] [Google Scholar]
13.World Health Organization. International Statistical Classification of Diseases and Related Health Problems, 10^th ed. rev. (ICD-10), 1992, Geneva: World Health Organization. [Google Scholar]
14.American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed., 1994, Washington DC: American Psychiatric Association. [Google Scholar]
15.Yokoyama A, Kato H, Yokoyama T, Igaki H, Tsujinaka T, Muto M, et al. Esophageal squamous cell carcinoma and aldehyde dehydrogenase-2 genotypes in Japanese females. Alcohol Clin Exp Res 2006; 30: 491–500. doi: 10.1111/j.1530-0277.2006.00053.x [DOI] [PubMed] [Google Scholar]
16.Harada S, Zhang S. New strategy for detection of ALDH2 mutant. Alcohol Alcohol 1993; 28 (Suppl): 11–3. doi: 10.1093/alcalc/28.supplement_1a.11 [DOI] [PubMed] [Google Scholar]
17.Xu Y, Carr LG, Bosron WF, Li TK, Edenberg HJ. Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification. Genomics 1998; 2: 209–14. [DOI] [PubMed] [Google Scholar]
18.Babor TF, Higgins-Biddle JC, Saunders JB, Monteiro MG. AUDIT: The Alcohol Use Disorders Identification Test, Guidelines for Use in Primary Care, Second Edition. 2001, World Health Organization. [Google Scholar]
19.Tan OT, Gaylarde PM, Sarkany I. Blocking of alcohol-induced flush with a combination of H1 and H2 histamine antagonists. The Lancet 1979; 2(8138): 365. doi: 10.1016/s0140-6736(79)90381-7 [DOI] [PubMed] [Google Scholar]
20.Tan OT, Stafford TJ, Sarkany I, Gaylarde PM, Tilsey C, Payne JP. Suppression of alcohol-induced flushing by a combination of H1 and H2 histamine antagonists. Br J Dermatol 1982; 107: 647–52. doi: 10.1111/j.1365-2133.1982.tb00522.x [DOI] [PubMed] [Google Scholar]
21.Miller NS, Goodwin DW, Jones FC, Gabrielli WF, Pardo MP, Anand MM, et al. Antihistamine blockade of alcohol-induced flushing in orientals. J Stud Alcohol 1988; 49: 16–20. doi: 10.15288/jsa.1988.49.16 [DOI] [PubMed] [Google Scholar]
22.Zimatkin SM, Anichtchik OV. Alcohol-histamine interactions. Alcohol Alcohol 1999; 34: 141–7. doi: 10.1093/alcalc/34.2.141 [DOI] [PubMed] [Google Scholar]
23.Truitt EB Jr, Gaynor CR, Mehl DL. Aspirin attenuation of alcohol-induced flushing and intoxication in Oriental and Occidental subjects. Alcohol Alcohol 1987; Supplment 1: 595–9. [PubMed] [Google Scholar]
24.Ho SB, DeMaster EG, Shafer RB, Levine AS, Morley JE, Go VL, et al. Opiate antagonist nalmefene inhibits ethanol-induced flushing in Asians: a preliminary study. Alcohol Clin Exp Res 1998; 12: 705–12. [DOI] [PubMed] [Google Scholar]
25.Yang SJ, Yokoyama A, Yokoyama T, Huang YC, Wu SY, Shao Y et al. Relationship between genetic polymorphisms of ALDH2 and ADH1B and esophageal cancer risk: A meta-analysis. World J Gastroenterol 2010; 16: 4210–20. doi: 10.3748/wjg.v16.i33.4210 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Du XY, Wen L, Hu YY, Deng SQ, Xie LC, Jiang GB et al. Association between the aldehyde dehydrogenase-2 rs671 G>A polymorphism and head and neck cancer susceptibility: A meta-analysis in East Asians. Alcohol Clin Exp Res 2021; 45: 307–17. [DOI] [PubMed] [Google Scholar]
27.Yokoyama A, Yokoyama T, Omori T. Past and current tendency for facial flushing after a small dose of alcohol is a marker for increased risk of upper aerodigestive tract cancer in Japanese drinkers. Cancer Sci 2010; 101: 2497–8. doi: 10.1111/j.1349-7006.2010.01709.x [DOI] [PubMed] [Google Scholar]
28.Higuchi S, Muramatsu T, Shigemori K, Saito M, Kono H, Dufour MC, et al. The relationship between low Km aldehyde dehydrogenase phenotype and drinking behavior in Japanese. J Stud Alcohol 1992: 53: 170–5. doi: 10.15288/jsa.1992.53.170 [DOI] [PubMed] [Google Scholar]
29.Higuchi S, Parrish KM, Dufour MC, Towle LH, Harford TC. The relationship between three subtypes of the flushing response and DSM-III alcohol abuse in Japanese. J Stud Alcohol 1992; 53: 553–60. doi: 10.15288/jsa.1992.53.553 [DOI] [PubMed] [Google Scholar]
30.Aoyama I, Ohashi S, Amanuma Y, Hirohashi K, Mizumoto A, Funakoshi M, et al. Establishment of a quick and highly accurate breath test for ALDH2 genotyping. Clin Trans Gastroenterol 2017; 8: e96. doi: 10.1038/ctg.2017.24 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Shin CM, Kim N, Cho SI, Sung J, Lee HJ. Validation of alcohol flushing questionnaires in determining inactive aldehyde dehydrogenase-2 and its clinical implication in alcohol-related diseases. Alcohol Clin Exp Res 2018; 42: 387–96. doi: 10.1111/acer.13569 [DOI] [PubMed] [Google Scholar]
32.Hsiao JR, Lee WT, Ou CY, Huang CC, Chang CC, Tsai ST, et al. Validation of alcohol flushing questionnaire to identify ALDH2 status in a case-control study of head and neck cancer. Alcohol Clin Exp Res 2019; 43: 1225–33. doi: 10.1111/acer.14049 [DOI] [PubMed] [Google Scholar]
33.Slutske WS, Heath AC, Madden PA, Bucholz KK, Dinwiddie SH, Dunne MP, et al. Is alcohol-related flushing a protective factor for alcoholism in Caucasians? Alcohol Clin Exp Res 1995; 19: 528–92. doi: 10.1111/j.1530-0277.1995.tb01552.x [DOI] [PubMed] [Google Scholar]
34.Wall TL, Thomasson HR, Ehlers CL. Investigator-observed alcohol-induced flushing but not self-report of flushing is a valid predictor of ALDH2 genotype. J Stud Alcohol 1996; 57: 267–72. doi: 10.15288/jsa.1996.57.267 [DOI] [PubMed] [Google Scholar]
35.Wall TL, Thomasson HR, Schuckit MA, Ehlers CL. Subjective feelings of alcohol intoxication in Asians with genetic variations of ALDH2 alleles. Alcohol Clin Exp Res 1992; 16: 991–5. doi: 10.1111/j.1530-0277.1992.tb01907.x [DOI] [PubMed] [Google Scholar]
36.Peng GS, Chen YC, Wang MF, Lai CL, Yin SJ. ALDH2*2 but not ADH1B*2 is a causative variant gene allele for Asian alcohol flushing after a low-dose challenge: correlation of the pharmacokinetic and pharmacodynamic findings. Pharmacogenet Genomics 2014; 24: 607–17. doi: 10.1097/FPC.0000000000000096 [DOI] [PubMed] [Google Scholar]
37.Yokoyama A, Kato H, Yokoyama T, Tsujinaka T, Muto M, Omori T, et al. Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma. Carcinogenesis 2002; 23: 1851–9. doi: 10.1093/carcin/23.11.1851 [DOI] [PubMed] [Google Scholar]
38.Ministry of Health, Labour and Welfare. Kokumin Eiyo no Genjo, 2001 (The National Nutrition Survey in Japan, 2001). 2003, pp 65. Daiichi Publishing Co, Tokyo, Japan: (in Japanese). [Google Scholar]
39.Health and Welfare Statistics Association. Kokumin Eisei no Doko, 2003 (Journal of Health and Welfare Statistics, 2003). 2003, vol 50, pp 49–50. The Association, Tokyo, Japan: (in Japanese). [Google Scholar]
40.Newman IM, Ding L, Shell DF, Lin L. How Social Reactions to Alcohol-Related Facial Flushing Are Affected by Gender, Relationship, and Drinking Purposes: Implications for Education to Reduce Aerodigestive Cancer Risks. Int J Environ Res Public Health 2017; 14: 622. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0255276.r001

Decision Letter 0

Nadine Bernhardt

9 Jun 2021

PONE-D-21-07074

Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

PLOS ONE

Dear Dr. Yokoyama,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Importantly pleae indicate more clearly which restrictions apply for your data sharing. PLOS journals require authors to make all data necessary to replicate their study’s findings publicly available without restriction at the time of publication. When specific legal or ethical restrictions prohibit public sharing of a data set, authors must indicate how others may obtain access to the data.

Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods. Additionally, you may consider deposition of data in a field relevant repository.

Once more we apologize for the delay in the evaluation process, but are confident that your manuscript can be accepted for publication when you have improved your data sharing and after you have implemented corrections as suggested by the reviewers.

Please submit your revised manuscript by Jul 24 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nadine Bernhardt, Ph.D.

Academic Editor

PLOS ONE

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

At this time, please address the following queries:

Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.
State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”
If any authors received a salary from any of your funders, please state which authors and which funders.
If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables should be uploaded) as separate "supporting information" files.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript examined ALDH2/ADH1B genotypes and alcohol flushing using a case-control study of alcohol use disorder (AUD) in over 5,000 Japanese men and women. Findings suggest that a two-item measure of past and current self-reported alcohol flushing was independently associated with AUD, over and above well-documented ALDH2 and ADH1B susceptibility genotypes.

Strengths of this study include its innovative approach disaggregating former flushing from ALDH2/ADH1B genotypes, use of both men and women, a relatively large sample size, and double-blind administration of the alcohol flushing questionnaire. Notwithstanding these strengths, there were also several significant concerns that dampened my enthusiasm for this project.

1. My most significant concern relates to potential control selection bias. First, individuals with AUD were not excluded from the control group. Although the authors estimate AUD rates may be low based on similar prior samples, the number of ‘AUD controls’ who have AUD and the impact of imperfect classification here ultimately remains unknown. Second, there was no data provided on alcohol use levels in controls. The ability to report on flushing and the hypothesized mechanisms of ALDH2/ADH1B on AUD all require alcohol consumption. If available, any data on alcohol use within controls would help estimate the strength of these concerns. Third, controls were borrowed from an esophageal cancer study. ALDH2/ADH1B genotypes are a known risk factor for esophageal cancer, such that it is less likely these cancer-free controls possessed certain genotypes. More attention to these limitations is necessary, with a much more nuanced and cautious interpretation of study findings.

2. The authors advocate for use of alcohol flushing data in addition to genotype to predict risk (rather than presence) of AUD. This was conceptually unclear and at times contradictory. The authors discuss how flushing might be present early on in individuals’ drinking histories and then disappear around the time habitual drinking emerges. They also discuss former flushing as a potential correlate of tolerance, a symptom of AUD. These interpretations seem to support former flushing as a correlate, rather than as a risk factor. Greater clarity is needed, especially because this could impact clinical implications (eg, whether flushing questions are useful to predict those who will go on to develop AUD or to use as screeners to detect AUD already present).

3. More information is needed to evaluate methodological quality of the alcohol flushing data: (i) Are data available on the measure’s reliability and validity? (ii) Has the self-reported questionnaire been correlated to observed flushing in the laboratory? (doi:10.15288/jsa.1996.57.267 notes differences in self-report and observed flushing) (iii) Have any psychometrics been reported in women? The authors do present some data on sensitivity/specificity in the limitations, but these are for detecting ALDH2 genotypes rather than capturing actual flushing response or flushing’s relation to AUD.

Additional concerns:

1. References are needed for lines 82-86.

2. It was unclear how the price of ALDH2/ADH1B genotyping is an argument in support of the current study (lines 86-88). The authors suggest genotype + flushing data better predicts AUD than flushing data alone, so no genotyping costs would be saved in this screening approach.

3. The introduction would benefit from discussing how knowledge about timing of flushing disappearance may help understand relations of alcohol flushing and ALDH2/ADH1B with AUD. Also, any prior findings on timing of flushing disappearance should be introduced.

4. Specify any a priori hypotheses in the final paragraph of the introduction.

5. There was a large amount of missing data on the temporal patterning of former flushing. This appears to be partly due to the item being added to assessments more recently. The authors should make this more explicit in the text if correct, report any other reasons for missing data, and explain how missing data were handled in analyses. This restricted sample may be less representative depending on the reasons for missing data, and this should be added as a study limitation. The authors could test and report any differences in study variables and demographics as a function of those with and without missing data to speak to generalizability concerns.

6. The authors should note in the text whether any part of this sample have been previously published on and, if so, include relevant citations and the need for these analyses.

7. The manuscript would benefit from an expanded discussion on what these findings mean for the broader literature on ALDH2/ADH1B relationships with alcohol flushing. Prior research has tended to use flushing questionnaires to infer ALDH2 genotype and risk for alcohol and cancer outcomes. The current findings suggest additional sources of variability in alcohol flushing aside from the ALDH2 and ADH1B genotypes tested. If alcohol flushing is not fully due to ALDH2/ADH1B, what implications does this have for interpreting existing literature, for using flushing questionnaires to infer genotypes, and for identifying AUD?

8. The discussion should mention limitations to the temporal patterning of former flushing data, in addition it the current discussion on the flushing questionnaire. There is potential recall bias for this retrospective self-report measure, with possible impacts on strength/direction of results.

9. There were several instances in which the text referred to men rather than men and women, and these should be revised as appropriate (line 100, line 238, Table 6 title).

10. Minor typo: The percentage listed on line 171 seems to contradict Table 1.

Reviewer #2: Alcohol-induced facial flushing and genetic polymorphisms of alcohol dehydrogenase (the fast-metabolizing ADH1B, rs122998) and aldehyde dehydrogenases (the inactive ALDH2, rs671), which enhance alcohol-induced flushing, are known protective factors for alcohol dependence (AD) in Asians. In this study, the authors examined whether combination of the flushing status and the 2 ADH1B/ALDH2 genotypes could better predict the risk of AD in Japanese men and women. Their study cohorts included a large number of male (3721) and a much smaller number female (335) AD cases and 610 male and 406 female historical community controls. The alcoholic and control subjects included current, former, and never flushing individuals as defined by simple questionnaires. They showed that never or former alcohol flushing, the slow-metabolizing ADH1B allele and the active ALDH2 genotype were risk factors for AD. In addition, never or former flushing and the ADH1B/ALDH2 genotype combinations were independent risk factors of AD in both men and women. Consistent with the finding that a large portion of the never or former flushing AD and control subjects were found to carry the inactive ALDH2 genotype, they also showed that non-flushing increased the OR of AD regardless of the ALDH2/ADH1B genetic polymorphisms. In a separate group of 66 former flushing AD, flushing response was shown to disappear around the start of alcohol misuse during early adulthood regardless of the the active and inactive ALDH2 genotype.

As the OR for the combination of the flushing status and the ALDH2/ADH1B genotype were lower than the OR for the flushing status alone or the genotypes alone, it is not clear how the combination can provide a better new strategy for AD risk assessment as claimed by the authors. Other than that, the study was well done and well written. There are no other major concerns. The following are some minor concerns and questions.

In line 171, the numbers 7.0% and 4.0% are different from those in Table 1.

Table 1 needs to show the p values for all the comparisons. For former flushing, was the differences significant between the AD and controls?

For Figure 1 and Figure 2, the different results as to the ADH1B genotype between males and females likely was due to a much small number of female subjects.

There is a discrepancy in the gender description of the subjects in Table 6. The title says it is for AD men. However, the Methods indicated that the 66 subjects include 61 men and 5 women.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jul 26;16(7):e0255276. doi: 10.1371/journal.pone.0255276.r002

Author response to Decision Letter 0

7 Jul 2021

Prof. Nadine Bernhardt

Academic Editor

PLOS ONE 　　　　　　　　　　　　　

Dear Prof. Bernhardt:

Ref: PONE-D-21-07074

We appreciate the comments of the reviewers. They were helpful in strengthening and clarifying portions of the manuscript.

Our point by point reply is included below:

According to the Editor’s suggestion, we share the large main data set because of its adequate anonymous nature.

Reply:　According to the reviewer’s comment, we added the following discussion.

Page 17, line 327: Another limitation was that alcohol dependence had not been excluded from the historical controls who had registered in case control studies of esophageal cancer as cancer-free controls between 2000 and 2001 (12,15,37). The proportion of control subjects who drank alcohol on 3 or more days per week and consumed at least 22 g of ethanol at a time was 56.0% in men (37) and 12.9% in women (15). These values were similar to the 53.3% in male adults and 9.1% in female adults reported by the National Nutrition Survey in 2001 (38). A 2003 nationwide survey showed a low prevalence (1.5% in men and 0.2% in women) of lifetime alcohol dependence diagnosed according to the ICD-10 criteria in Japanese adults (11). The mortality rate associated with esophageal cancer per 100,000 in 2001 was 10.5 in men and 1.3 in women (39). Thus, although the ADH1B and ALDH2 genotypes are risk factors for both alcohol dependence (6-10) and esophageal cancer (15,25,37), it is unlikely that the potential selection bias of the controls, i.e., esophageal-cancer-free and imperfect exclusion of alcohol dependence, significantly affected the present results due to their low prevalence.

Reply: We discussed these issues as follows.

Page 15, line 260: Tolerance against alcohol flushing may be acquired due to a pattern of heavy alcohol use in some subjects during their young adulthood, and disappearance of flushing in some former flushers could have been a consequence of alcohol dependence rather than a risk factor for alcohol dependence. It would be more relevant to study detailed drinking behavior during the young adulthood. However, the flushing tendency disappeared before the start of habitual drinking in 45.5% of former flushers, and this interpretation was not applicable to never flushers among inactive ALDH2*1/*2 carriers. The mean age until which the flushing tendency continued and the mean age at the first hospital visit for alcohol-related problems were 25.7 years and 51.4 years, respectively. Thus, use of alcohol flushing data in addition to ALDH2 genotyping is useful to predict, and could be expected to reduce, the risk of future development of alcohol dependence, especially in younger generations. Combined use of this information with established screening tools, e.g., Alcohol Use Disorders Identification Test (AUDIT; 18), may be more useful to detect alcohol use disorders that are already present.

Reply: We discussed these issues as follows.

Page 17, line 322: An oral alcohol challenge test using 0.56 g ethanol/kg showed lower specificity of self-reported flushing for predicting the presence of inactive ALDH2 in comparison to investigator-observed flushing (34). Self-reported flushing may be a net self-evaluation of the perception of general discomfort (35) and cardiovascular effects (36) in flushing responses.

Additional concerns:

1. References are needed for lines 82-86.

Reply: We added the reference (6).

Reply: We changed the sentence as follows.

Page 5, line 77: ALDH2 plus ADH1B genotyping using buccal smear DNA is commercially available and inexpensive in Japan.

Reply: According to the reviewer’ comment, we added the following paragraph before the final paragraph of the introduction.

Page 6, line 97: According to a report, alcohol consumption in inactive ALDH2*1/*2 carriers in the general population increased in the order of current flushers, former flushers, and never flushers (12). If the flushing disappearance occurred in young adulthood and not around the time of development of alcohol dependence in many former flushers who are alcohol-dependent, it is conceivable that both former flushing and never flushing may serve as independent risk factors for alcohol dependence, in combination with the ADH1B and ALDH2 genotypes.

Reply: To clarify that data of the temporal patterning of former flushing were representative data in the population, we added how the data were corrected in the methods.

Page 9, line 135: Between July 2019 and December 2020, 283 Japanese subjects with alcohol dependence (253 men and 30 women) aged 40–79 years who visited the Center for the first time were asked about their alcohol flushing history and underwent routine ADH1B and ALDH2 genotyping. Of the subjects, 7 (6 men and one women), 69 (64 men and 5 women), and 207 (183 men and 24 women) reported themselves as current flushers, former flushers, and never flushers, respectively. We asked 66 former flushers (61 men and 5 women) the following question: “Up until what age did you have a tendency to develop facial flushing immediately after drinking a glass of beer?”

6. The authors should note in the text whether any part of this sample have been previously published on and, if so, include relevant citations and the need for these analyses.

Reply: According to the comment, we added the following sentence in the methods.

Page 7, line 119: Male subjects with alcohol dependence were a subgroup (40-79 years) of 4051 subjects aged 30-79 years, in which we recently reported the associations between the results of an alcohol flushing questionnaire and the ALDH1B and ALDH2 genotypes (6).

Reply: We discussed these issue as follows in the discussion.

Page 16, line 288: Inactive ALDH2 has been reported as a strong risk factor for squamous cell carcinoma of the upper aerodigestive tract in East Asian drinkers (25,26). Current or former flushing has been shown to be a predictor of the risk of cancer as a surrogate marker of inactive ALDH2 in various drinking populations (27). However, the cancer risk in the current or former flushers was substantially lower than the risk in ALDH2*1/*2 carriers in an alcohol-dependent population overall (27). This could be partly attributable to the existence of additional causes of alcohol flushing besides the ALDH2 genotype in persons susceptible to alcohol dependence. Investigation is warranted, in a future study, of whether and how the combination of alcohol flushing status and the ALDH2 plus ADH1B genotype might affect drinking behaviors and manifestations of alcohol-related problems.

Reply: We discussed these issues as follows in the discussion.

Page 18, line 345: In addition to the retrospective self-reported alcohol flushing questionnaire, the temporal pattern of former flushing also has potential recall bias. Furthermore, it was evaluated only in male subjects with alcohol dependence, which may differ from the case in the general population and women. There is a higher probability of Chinese university students thinking that they should encourage a flusher to stop drinking or drink less when the flusher is a woman (40), which may prevent the development of tolerance in female flushers.

9. There were several instances in which the text referred to men rather than men and women, and these should be revised as appropriate (line 100, line 238, Table 6 title).

Reply: According this comments, we checked and correct the text appropriately.

10. Minor typo: The percentage listed on line 171 seems to contradict Table 1.

Reply:　We corrected the percentage.

Reply: To clarify these issues, we added the following discussion.

Page 14, line 243: Current flushers were less frequently reported in the subject groups with alcohol dependence than in the control group, and never flushers and former flushers showed similarly elevated ORs for alcohol dependence as compared to current flushers, among both men and women. However, the flushing status subdivided subjects with each of the ALDH2 and ADH1B genotypes into groups with substantially lower and higher ORs. Never or former flushing markedly increased the ORs of alcohol dependence among carriers of any ALDH2 and ADH1B genotype combination, compared with current flushing. Commercially available ALDH2 plus ADH1B genotyping has been used as a preventive tool against alcohol dependence in Japan. Combined evaluation of the alcohol flushing status and the ALDH2 plus ADH1B genotype may show superior ability than that of either alone for predicting the risk of alcohol dependence.

The following are some minor concerns and questions.

In line 171, the numbers 7.0% and 4.0% are different from those in Table 1.

Reply: We corrected the numbers correctly.

Table 1 needs to show the p values for all the comparisons. For former flushing, was the differences significant between the AD and controls?

Reply: We added the results of comparing the prevalence of each category between alcohol dependence and controls in the table 1.

For Figure 1 and Figure 2, the different results as to the ADH1B genotype between males and females likely was due to a much small number of female subjects.

Reply: We need to evaluate the possible gender difference and discussed this issue as follows.

Page 18, line 347: Furthermore, it was evaluated only in male subjects with alcohol dependence, which may differ from the case in the general population and women. There is a higher probability of Chinese university students thinking that they should encourage a flusher to stop drinking or drink less when the flusher is a woman (40), which may prevent the development of tolerance in female flushers. Generalization of the results obtained in this study, which was based on investigations of treatment-seeking alcohol-dependent patients, will require confirmation in various heavy-drinking populations, including groups with mild alcohol-dependent cases and a large number female cases, as well as in large control populations.

There is a discrepancy in the gender description of the subjects in Table 6. The title says it is for AD men. However, the Methods indicated that the 66 subjects include 61 men and 5 women.

Reply: We corrected the title appropriately.

Attachment

Submitted filename: PONE PPR2021CC.docx

Click here for additional data file.^{(24.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0255276.r003

Decision Letter 1

Nadine Bernhardt

14 Jul 2021

Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

PONE-D-21-07074R1

Dear Dr. Yokoyama,

We appreciate the effort you have taken to address all issues raised during the revision process and thus we’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nadine Bernhardt, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors were generally responsive to my comments, and my opinions on the strengths of this study remain the same.

My most significant prior concern was potential control selection bias. The authors added data on alcohol use within controls, which was generally comparable to a past national survey, and I think this strengthens interpretability of the results. Although the authors are still limited by available data such that misclassification (AUD in controls) and uneven exposure (controls who have not consumed alcohol) remain unknown, I appreciate their work estimating comparability and willingness to speak directly to these challenges in the text.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0255276.r004

Acceptance letter

Nadine Bernhardt

16 Jul 2021

PONE-D-21-07074R1

Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

Dear Dr. Yokoyama:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Nadine Bernhardt

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data

(XLSX)

Click here for additional data file.^{(142.7KB, xlsx)}

Attachment

Submitted filename: PONE PPR2021CC.docx

Click here for additional data file.^{(24.4KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

[pone.0255276.ref001] 1.Takeshita T, Mao XQ, Morimoto K. The contribution of polymorphism in the alcohol dehydrogenase β subunit to alcohol sensitivity in a Japanese population. Hum Genet 1996; 97: 409–13. doi: 10.1007/BF02267057 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref002] 2.Chen WJ, Chen CC, Yu JM, Cheng AT. Self-reported flushing and genotypes of ALDH2, ADH2, and ADH3 among Taiwanese Han. Alcohol Clin Exp Res 1998; 22: 1048–52. [PubMed] [Google Scholar]

[pone.0255276.ref003] 3.Matsuo K, Wakai K, Hirose K, Saito T, Tajima K. Alcohol dehydrogenase 2 His47Arg polymorphism influences drinking habit independently of aldehyde dehydrogenase 2 Glu487Lys polymorphism: analysis of 2,299 Japanese subjects. Cancer Epidemiol Biomarkers Prev 2006; 15: 1009–13. doi: 10.1158/1055-9965.EPI-05-0911 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref004] 4.Luczak SE, Pandika D, Shea SH, Eng MY, Liang T, Wall TL. ALDH2 and ADH1B interactions in retrospective reports of low-dose reactions and initial sensitivity to alcohol in Asian American college students. Alcohol Clin Exp Res 2011; 35: 1238–45. doi: 10.1111/j.1530-0277.2011.01458.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255276.ref005] 5.Hayashida M, Kamada Y, Ota T, Kojima S, Iwao-Koizumi K, Murata S, et al. Associations between ALDH2 and ADH1B Genotypes and Ethanol-Induced Cutaneous Erythema in Young Japanese Women. Nihon Eiseigaku Zasshi 2015; 70: 134–8 (in Japanese with English abstract). [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref006] 6.Yokoyama A, Yokoyama T, Matsui T, Mizukami T, Kimura M, Matsushita S, et al. Impacts of interactions between ADH1B and ALDH2 genotypes on alcohol flushing, alcohol reeking on the day after drinking, and age distribution in Japanese alcohol-dependent men. Pharmacogenet Genomics 2020; 30: 54–60. doi: 10.1097/FPC.0000000000000395 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref007] 7.Higuchi S, Matsushita S, Muramatsu T, Kinoshita T, Takagi S, Kono H. Alcohol and aldehyde dehydrogenase polymorphism and the risk for alcoholism. Am J Psychiatry 1995. 152, 1219–1221. doi: 10.1176/ajp.152.8.1219 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref008] 8.Chen CC, Lu RB, Chen YC, Wang MF, Chang YC, Li TK, et al. Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism. Am J Hum Genet 1999; 65: 795–807. doi: 10.1086/302540 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255276.ref009] 9.Luczak SE, Glatt SJ, Wall TL. Meta-analyses of ALDH2 and ADH1B with alcohol dependence in Asians. Psychol Bull 2006; 132: 607–21. doi: 10.1037/0033-2909.132.4.607 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref010] 10.Kim DJ, Choi IG, Park BL, Lee BC, Ham BJ, Yoon S, et al. Major genetic components underlying alcoholism in Korean population. Hum Mole Genetics 2008; 17: 854–8. doi: 10.1093/hmg/ddm357 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref011] 11.Osaki Y, Kinjo A, Higuchi S, Matsumoto H, Yuzuriha T, Horie Y, et al. Prevalence and trends in alcohol dependence and alcohol use disorders in Japanese adults; Results from periodical nationwide surveys. Alcohol Alcohol 2016; 51: 465–73. doi: 10.1093/alcalc/agw002 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref012] 12.Yokoyama T, Yokoyama A, Kato H, Tsujinaka T, Muto M, Omori T, et al. Alcohol flushing, alcohol and aldehyde dehydrogenase genotypes, and risk for esophageal squamous cell carcinoma in Japanese men. Cancer Epidemiol Biomarkers Prev 2003; 12: 1227–33. [PubMed] [Google Scholar]

[pone.0255276.ref013] 13.World Health Organization. International Statistical Classification of Diseases and Related Health Problems, 10^th ed. rev. (ICD-10), 1992, Geneva: World Health Organization. [Google Scholar]

[pone.0255276.ref014] 14.American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed., 1994, Washington DC: American Psychiatric Association. [Google Scholar]

[pone.0255276.ref015] 15.Yokoyama A, Kato H, Yokoyama T, Igaki H, Tsujinaka T, Muto M, et al. Esophageal squamous cell carcinoma and aldehyde dehydrogenase-2 genotypes in Japanese females. Alcohol Clin Exp Res 2006; 30: 491–500. doi: 10.1111/j.1530-0277.2006.00053.x [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref016] 16.Harada S, Zhang S. New strategy for detection of ALDH2 mutant. Alcohol Alcohol 1993; 28 (Suppl): 11–3. doi: 10.1093/alcalc/28.supplement_1a.11 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref017] 17.Xu Y, Carr LG, Bosron WF, Li TK, Edenberg HJ. Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification. Genomics 1998; 2: 209–14. [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref018] 18.Babor TF, Higgins-Biddle JC, Saunders JB, Monteiro MG. AUDIT: The Alcohol Use Disorders Identification Test, Guidelines for Use in Primary Care, Second Edition. 2001, World Health Organization. [Google Scholar]

[pone.0255276.ref019] 19.Tan OT, Gaylarde PM, Sarkany I. Blocking of alcohol-induced flush with a combination of H1 and H2 histamine antagonists. The Lancet 1979; 2(8138): 365. doi: 10.1016/s0140-6736(79)90381-7 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref020] 20.Tan OT, Stafford TJ, Sarkany I, Gaylarde PM, Tilsey C, Payne JP. Suppression of alcohol-induced flushing by a combination of H1 and H2 histamine antagonists. Br J Dermatol 1982; 107: 647–52. doi: 10.1111/j.1365-2133.1982.tb00522.x [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref021] 21.Miller NS, Goodwin DW, Jones FC, Gabrielli WF, Pardo MP, Anand MM, et al. Antihistamine blockade of alcohol-induced flushing in orientals. J Stud Alcohol 1988; 49: 16–20. doi: 10.15288/jsa.1988.49.16 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref022] 22.Zimatkin SM, Anichtchik OV. Alcohol-histamine interactions. Alcohol Alcohol 1999; 34: 141–7. doi: 10.1093/alcalc/34.2.141 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref023] 23.Truitt EB Jr, Gaynor CR, Mehl DL. Aspirin attenuation of alcohol-induced flushing and intoxication in Oriental and Occidental subjects. Alcohol Alcohol 1987; Supplment 1: 595–9. [PubMed] [Google Scholar]

[pone.0255276.ref024] 24.Ho SB, DeMaster EG, Shafer RB, Levine AS, Morley JE, Go VL, et al. Opiate antagonist nalmefene inhibits ethanol-induced flushing in Asians: a preliminary study. Alcohol Clin Exp Res 1998; 12: 705–12. [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref025] 25.Yang SJ, Yokoyama A, Yokoyama T, Huang YC, Wu SY, Shao Y et al. Relationship between genetic polymorphisms of ALDH2 and ADH1B and esophageal cancer risk: A meta-analysis. World J Gastroenterol 2010; 16: 4210–20. doi: 10.3748/wjg.v16.i33.4210 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255276.ref026] 26.Du XY, Wen L, Hu YY, Deng SQ, Xie LC, Jiang GB et al. Association between the aldehyde dehydrogenase-2 rs671 G>A polymorphism and head and neck cancer susceptibility: A meta-analysis in East Asians. Alcohol Clin Exp Res 2021; 45: 307–17. [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref027] 27.Yokoyama A, Yokoyama T, Omori T. Past and current tendency for facial flushing after a small dose of alcohol is a marker for increased risk of upper aerodigestive tract cancer in Japanese drinkers. Cancer Sci 2010; 101: 2497–8. doi: 10.1111/j.1349-7006.2010.01709.x [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref028] 28.Higuchi S, Muramatsu T, Shigemori K, Saito M, Kono H, Dufour MC, et al. The relationship between low Km aldehyde dehydrogenase phenotype and drinking behavior in Japanese. J Stud Alcohol 1992: 53: 170–5. doi: 10.15288/jsa.1992.53.170 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref029] 29.Higuchi S, Parrish KM, Dufour MC, Towle LH, Harford TC. The relationship between three subtypes of the flushing response and DSM-III alcohol abuse in Japanese. J Stud Alcohol 1992; 53: 553–60. doi: 10.15288/jsa.1992.53.553 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref030] 30.Aoyama I, Ohashi S, Amanuma Y, Hirohashi K, Mizumoto A, Funakoshi M, et al. Establishment of a quick and highly accurate breath test for ALDH2 genotyping. Clin Trans Gastroenterol 2017; 8: e96. doi: 10.1038/ctg.2017.24 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0255276.ref031] 31.Shin CM, Kim N, Cho SI, Sung J, Lee HJ. Validation of alcohol flushing questionnaires in determining inactive aldehyde dehydrogenase-2 and its clinical implication in alcohol-related diseases. Alcohol Clin Exp Res 2018; 42: 387–96. doi: 10.1111/acer.13569 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref032] 32.Hsiao JR, Lee WT, Ou CY, Huang CC, Chang CC, Tsai ST, et al. Validation of alcohol flushing questionnaire to identify ALDH2 status in a case-control study of head and neck cancer. Alcohol Clin Exp Res 2019; 43: 1225–33. doi: 10.1111/acer.14049 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref033] 33.Slutske WS, Heath AC, Madden PA, Bucholz KK, Dinwiddie SH, Dunne MP, et al. Is alcohol-related flushing a protective factor for alcoholism in Caucasians? Alcohol Clin Exp Res 1995; 19: 528–92. doi: 10.1111/j.1530-0277.1995.tb01552.x [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref034] 34.Wall TL, Thomasson HR, Ehlers CL. Investigator-observed alcohol-induced flushing but not self-report of flushing is a valid predictor of ALDH2 genotype. J Stud Alcohol 1996; 57: 267–72. doi: 10.15288/jsa.1996.57.267 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref035] 35.Wall TL, Thomasson HR, Schuckit MA, Ehlers CL. Subjective feelings of alcohol intoxication in Asians with genetic variations of ALDH2 alleles. Alcohol Clin Exp Res 1992; 16: 991–5. doi: 10.1111/j.1530-0277.1992.tb01907.x [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref036] 36.Peng GS, Chen YC, Wang MF, Lai CL, Yin SJ. ALDH2*2 but not ADH1B*2 is a causative variant gene allele for Asian alcohol flushing after a low-dose challenge: correlation of the pharmacokinetic and pharmacodynamic findings. Pharmacogenet Genomics 2014; 24: 607–17. doi: 10.1097/FPC.0000000000000096 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref037] 37.Yokoyama A, Kato H, Yokoyama T, Tsujinaka T, Muto M, Omori T, et al. Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma. Carcinogenesis 2002; 23: 1851–9. doi: 10.1093/carcin/23.11.1851 [DOI] [PubMed] [Google Scholar]

[pone.0255276.ref038] 38.Ministry of Health, Labour and Welfare. Kokumin Eiyo no Genjo, 2001 (The National Nutrition Survey in Japan, 2001). 2003, pp 65. Daiichi Publishing Co, Tokyo, Japan: (in Japanese). [Google Scholar]

[pone.0255276.ref039] 39.Health and Welfare Statistics Association. Kokumin Eisei no Doko, 2003 (Journal of Health and Welfare Statistics, 2003). 2003, vol 50, pp 49–50. The Association, Tokyo, Japan: (in Japanese). [Google Scholar]

[pone.0255276.ref040] 40.Newman IM, Ding L, Shell DF, Lin L. How Social Reactions to Alcohol-Related Facial Flushing Are Affected by Gender, Relationship, and Drinking Purposes: Implications for Education to Reduce Aerodigestive Cancer Risks. Int J Environ Res Public Health 2017; 14: 622. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Combinations of alcohol-induced flushing with genetic polymorphisms of alcohol and aldehyde dehydrogenases and the risk of alcohol dependence in Japanese men and women

Akira Yokoyama

Tetsuji Yokoyama

Mitsuru Kimura

Sachio Matsushita

Masako Yokoyama

Roles

Abstract

Objective

Methods

Results

Conclusion

Introduction

Materials and methods

Subjects

The simple flushing questionnaire

ALDH2 and ADH1B genotypes

Statistical analyses

Results

Table 1. Alcohol flushing and ADH1B and ALDH2 genotypes in the male and female subjects.

Fig 1. Comparison of the alcohol flushing between alcohol-dependent men and controls according to the combinations of ALDH2 and ADH1B genotypes.

Fig 2. Comparison of the alcohol flushing between alcohol-dependent women and controls according to the combinations of ALDH2 and ADH1B genotypes.

Table 2. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to alcohol flushing status and/or the combinations of ALDH2 and ADH1B genotypes in the male subjects.

Table 3. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to alcohol flushing status and/or the combinations of ALDH2 and ADH1B genotypes in the female subjects.

Table 4. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to the combinations of alcohol flushing status and ALDH2 and ADH1B genotypes in the male subjects.

Table 5. Age-adjusted odds ratios (95% confidence intervals) of alcohol dependence according to the combinations of alcohol flushing status and ALDH2 and ADH1B genotypes in the female subjects.

Table 6. Temporal profiles for drinking and flushing in former-flushing, alcohol-dependent subjects according to ALDH2 genotype.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Nadine Bernhardt

Roles

Author response to Decision Letter 0

Decision Letter 1

Nadine Bernhardt

Roles

Acceptance letter

Nadine Bernhardt

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases