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

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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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.

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Reviewer #1: No

Reviewer #2: No

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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.

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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

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2. 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: Yes

Reviewer #2: Yes

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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?

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: Yes

Reviewer #2: Yes

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5. 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

**********

6. 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: 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)

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Reviewer #1: No

Reviewer #2: No