Alopecia areata is not a risk factor for heart diseases: A 10-year retrospective cohort study.

Alopecia areata (AA) is an autoimmune skin disease caused by chronic inflammation of hair follicles. Chronic inflammatory skin diseases such as psoriasis and lupus erythematosus can increase the risk of cardiovascular diseases. However, the relationship between AA and heart diseases (HDs) remains unclear. Therefore, we conducted this retrospective cohort study to evaluate the risk of subsequent HDs in patients with AA. We reviewed 3770 cases of AA and from 18,850 age, sex, and income level-matched controls from the National Health Insurance Service-National Sample Cohort. In the subgroup analysis, patients who suffered from alopecia totalis, alopecia universalis, and ophiasis were designated as patients with severe AA and patients having the disease for over a year were designated as patients with long-standing AA. As a result, we found that AA was not associated with a higher risk of heart failure, angina pectoris, or myocardial infarction. There was no significant increase in the risk of overall HD associated with AA (adjusted hazard ratio: 1.17; 95% confidence interval: 0.93-1.48; p = 0.177). Neither the severity nor the duration of AA was related to an increased risk of HDs. During the study period, AA patients did not show a significantly higher cumulative incidence of HDs than controls (log-rank p = 0.157). In conclusion, AA does not increase the risk of HD.

Introduction

Alopecia areata (AA) is an autoimmune disorder that results in non-scarring hair loss due to an inflammatory response in the hair follicles. Particularly, body attacking its own hair bulbs through CD8+ T lymphocytes is thought to be the primary cause of the disease [1]. Recent studies have revealed that AA is related to autoimmune diseases mediated by helper T cells such as atopic dermatitis, lupus erythematosus, and thyroid diseases [2-7].

Patients with AA show various degrees of involvement including generally well-demarcated patchy loss in AA, loss of all hair on the scalp in alopecia totalis (AT), and complete loss of hair on the scalp as well as on the body in alopecia universalis (AU) [8]. As AA involves a considerable area of the affected body parts, AA patients experience lowered self-esteem and consequently, suffer from various psychiatric problems [9,10]. As AA is related to many other immunologic disorders and is directly connected to patients’ quality of life, AA and its comorbidities have garnered nationwide attention.

Increasing evidence suggests that chronic skin inflammatory diseases like atopic dermatitis, psoriasis, and vitiligo are associated with an increased risk of cardiovascular disease [11,12]. In this context, we sought to clarify whether AA patients have a higher risk of cardiac diseases such as ischemic heart disease and heart failure. Some studies claim that AA patients are vulnerable to cardiovascular diseases [13,14]. As AA patients showed increased cardiac marker, we postulated that T cells affecting hair bulbs might extend to other systems. However, Huang et al. [8] suggested that AA patients had decreased risk for stroke and acute myocardial infarction, though the results were not statistically significant. We postulated that such differences could originate from other factors such as racial differences. Sensitivity to the metabolic diseases can vary according to patients’ racial and ethnic characteristics because of their cultural and genetic differences [15,16]. It is necessary to evaluate the risk of cardiac disease in Asian patients with AA.

We conducted the present retrospective cohort study using administrative healthcare data. The objective of this study was to determine the risk of heart diseases (HDs) in Korean AA patients.

Methods

1. Data source

The present study was conducted using data from the Korean National Health Insurance Service (NHIS)-National Sample Cohort from 2002 to 2013 [17]. A total of 1,125,691 participants were randomly selected from 97.1% of the Korean population enrolled in the NHIS and were followed up for 12 years. The diagnosis of the cohort was based on the International Classification of Diseases, Tenth Revision (ICD-10). The data consisted of encrypted personal information, diagnostic codes, prescribed drugs, and medical costs. The validity and the usefulness of the cohort data has been verified in several studies [10,18,19].

2. Study population and covariates

From the sample cohort, subjects with ICD-10 codes for AA (L63 and all its sub-classification codes including AT [L63.0], AU [L63.1], ophiasis [L63.2], other AA [L63.8], and unspecified AA [L63.9]) were included in the AA cohort. Cohort entry date was defined as the day when the patients received these codes for the first time. To obtain a control group, age, cohort entry date, sex, and income level-matched random samples who had not been diagnosed with AA codes were included (1:5 ratio of patients and controls).

The study subjects were followed up until they were diagnosed with HDs such as heart failure (HF), angina pectoris (AP), acute myocardial infarction (AMI), and chronic myocardial infarction (CMI) with the corresponding ICD-10 codes (HF [I50 and its subclassification codes], AP [I20 and its subclassification codes], AMI [I21, I22, I23, and I24 and its subclassification codes], CMI [I25 and its subclassification codes])during the observation period (from January 2004 to December 2013). Subjects who did not develop HDs were followed up until death, emigration, or the end of December 2013, whichever event occurred first. For accuracy, we selected patients who had these diagnostic codes assigned only by dermatologists in case of AA and by internists in case of HDs. Subjects who had HD codes before receiving any AA code, who had AA codes during the washout period (from 2002 to 2003), or those aged less than 40 years were excluded. Subjects with systemic comorbidities such as hypertension, diabetes, and dyslipidemia before the cohort entry date were identified. Additionally, HD-related comorbidities including stress disorders, peripheral vascular diseases, atherosclerosis, and stroke were also assessed due to their potential as confounders. In subgroup analyses, we further defined severe AA group as patients with AT, AU, or ophiasis diagnostic codes (other AA patients were categorized into mild AA group). We also designated a long-standing AA group, which was defined as patients with AA for over a year with more than ten outpatient visits (other AA patients were categorized into episodic AA group). A schematized flow chart of the study is shown in Fig 1.

Fig 1

Study flowchart.

AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; HD, heart disease. *AA codes: L63 and its sub-classified codes. †HD codes: I20 (angina), I21-I25 (myocardial infarction), I50 (heart failure).

3. Statistical analysis

Initially, we used the chi-square test to categorize and to evaluate the statistical significance of the data as a descriptive analysis. After adjusting for significant differences, we obtained hazard ratios (HRs) and 95% confidence intervals (CIs) by Cox proportional hazard regression analysis. Only the results with a two-tailed p-value less than 0.05 were considered significant. In addition, the Kaplan-Meier method was used to estimate the risk of HD. Statistical analyses were performed using SAS 9.2 (SAS Institute Inc., Cary, NC, USA) and MedCalc Statistical Software 18.9.1 (MedCalc Software Ltd., Ostend, Belgium).

4. Ethical statement

The present study used de-identified data and was approved by the Institutional Review Board of the Ajou University Hospital (IRB-No: AJIRB-MED-EXP-18-404).

Results

1. Demographic and clinical characteristics of the study population

During the observation period, 3770 AA patients and 18,850 matched controls (a ratio of 1:5) were selected from the sample cohort of 1,125,691 participants. The prevalence of dyslipidemia was significantly higher in the AA group when compared with the control group. However, there were no significant differences in other comorbidities between the two groups (Table 1).

Table 1

Demographic and clinical characteristics of the study population.

Characteristics	AA patients	Control subjects	P value
Case No.	3770	18850
Sex, N (%)			1.000
Men	1662 (44.1)	8310 (44.1)
Women	2108 (55.9)	10540 (55.9)
Age distribution, N (%)			1.000
40–59	3283 (87.1)	16415 (87.1)
60–79	478 (12.7)	2390 (12.7)
80-	9 (0.2)	45 (0.2)
Income level, N (%)			1.000
Lower third	778 (20.6)	3890 (20.6)
Middle third	1187 (31.5)	5935 (31.5)
Upper third	1805 (47.9)	9025 (47.9)
Common systemic comorbidities, N (%)
Hypertension	574 (15.2)	2959 (15.7)	0.476
Diabetes	224 (5.9)	1138 (6.0)	0.849
Dyslipidemia	169 (4.5)	676 (3.6)	0.009
Other HD related comorbidites, N (%)
Stress disorder	25 (0.7)	90 (0.5)	0.163
Peripheral vascular diseases and atherosclerosis	163 (0.9)	40 (1.1)	0.255
Stroke	87 (2.3)	402 (2.1)	0.503

HD, heart disease.

Bolding indicates statistical significance.

2. Incidence of HD in AA patients and control subjects

The most common HD comorbidity was AP, followed by CMI, HF, and AMI in both groups. In the univariate analysis, the HRs for HF, AP, and CMI were higher in the AA group when compared with the control subjects. However, the result was not statistically significant. After adjusting for confounders, the HR remained insignificant.

The highest HR was found for HF (HR: 1.75; 95% CI: 0.93–3.29) in the univariate analysis. After adjustment, the highest HR was observed for HF (HR: 1.74; 95% CI: 0.93–3.28). However, both of these results were not statistically significant.

During the follow-up period, the overall incidence of HDs in the control and in the AA groups was 1.99% and 2.36%, respectively. The risk of developing at least one HD was higher in the AA group, but the difference was not significant (adjusted HR: 1.17; 95% CI: 0.93–1.48) (Table 2).

Table 2

Hazard ratio and 95% confidence intervals of heart diseases in patients with alopecia areata and in control subjects.

		Univariate			Adjusted*
Heart diseases comorbidities	Event/total	HR (95% CI)		P-value	HR (95% CI)		P-value
Heart failure
Control subjects	37/18850	1.00	(reference)		1.00	(reference)
AA patients	13/3770	1.75	(0.93–3.29)	0.083	1.74	(0.93–3.28)	0.084
Angina pectoris
Control subjects	264/18850	1.00	(reference)		1.00	(reference)
AA patients	62/3770	1.17	(0.89–1.54)	0.269	1.16	(0.88–1.53)	0.300
Acute myocardial infarction
Control subjects	45/18850	1.00	(reference)		1.00	(reference)
AA patients	4/3770	0.44	(0.16–1.23)	0.118	0.45	(0.16–1.24)	0.121
Chronic myocardial infarction
Control subjects	79/18850	1.00	(reference)		1.00	(reference)
AA patients	17/3770	1.07	(0.63–1.81)	0.797	1.07	(0.63–1.80)	0.804
†All the heart disease occurrence
Control subjects	375/18850	1.00	(reference)		1.00	(reference)
AA patients	89/3770	1.18	(0.94–1.49)	0.158	1.17	(0.93–1.48)	0.177

AA, alopecia areata; CI, confidence interval.

*Adjusted for the variables of significant difference in frequency by Chi square test.

†All patients who developed at least one heart diseases in the table.

3. Cumulative incidence of HD in patients with AA

The Kaplan-Meier curve was plotted to determine the cumulative incidence of HDs in patients with AA and in control subjects. Patients with AA had an increased risk for HDs, although the difference was not statistically significant (log-rank p-value = 0.157) (Fig 2).

Fig 2

Cumulative incidence of heart diseases in patients with alopecia areata.

The difference between the two study cohorts was calculated using the log-rank test.

4. Subgroup analysis according to disease severity and duration

Among the 3770 patients, 106 were patients with severe AA who had AT, AU, or ophiasis and 419 patients had long-standing AA. In the subgroup analysis based on the disease severity, no HD occurrence was observed in patients with severe AA. However, HDs occurred in 2.43% of the patients with mild AA. Based on the disease duration, the occurrence of HD events in long-standing AA patients and in episodic AA patients was 2.15% and 2.39%, respectively. The HRs for the HDs in patients with mild AA and in patients with episodic AA were higher than those in matched controls. However, the difference was not significant (adjusted HR: 1.21; 95% CI: 0.96–1.52 for the mild AA subgroup, adjusted HR: 1.22; 95% CI: 0.96–1.55 for the episodic AA subgroup). HR for HDs in the long-standing AA subgroup was lower than that in the control group, but the difference was not significant (adjusted HR 0.89; 95% CI: 0.46–1.73) (Table 3).

Table 3

Hazard ratio and 95% confidence intervals of heart diseases according to the subgroup analysis.

Subgroup analysis was performed according to disease severity and duration.

		Univariate			Adjusted*
	Event/total	HR (95% CI)		P-value	HR (95% CI)		P-value
By disease severity
Control subjects	375/18850	1.00	(reference)		1.00	(reference)
Mild AA patients	89/3664	1.27	(0.97–1.53)	0.096	1.21	(0.96–1.52)	0.110
Severe AA patients	0/106	NA	NA	NA	NA	NA	NA
By disease duration
Control subjects	375/18850	1.00	(reference)		1.00	(reference)
Episodic AA patients	80/3342	1.23	(0.96–1.56)	0.099	1.22	(0.96–1.55)	0.113
Long-standing AA patients	9/419	0.90	(0.46–1.74)	0.746	0.89	(0.46–1.73)	0.738

AA, alopecia areata; CI, confidence interval; NA, not applicable

*Adjusted for the variables of significant difference in frequency by Chi square test.

Discussion

In this retrospective cohort study, we found that AA was not related to an increased risk of HD. This is the first study to include such a large cohort of Asian patients with AA to assess the risk for HDs. Recently, a study involving heterogeneous AA population in the USA found that AA was not related to an increased risk of AMI. The results were not statistically significant (odds ratio: 0.91; 95% CI: 0.59–1.39) [8]. Though there was a difference in the racial demographic characteristics between the aforementioned study and the present study, there was no discrepancy in the finding that AA patients were not prone to develop HDs. Therefore, it seems that racial differences do not affect the risk of HD.

A causal association between AA and HDs requires the presence of a consistent association in different studies, a dose-response relationship, and biologically plausible mechanisms. However, studies evaluating the risk of HDs in AA patients have not shown such consistency.

C3H/HeJ mice with AA had higher levels of cardiac troponin, a serum HD marker, and showed a higher risk of cardiac hypertrophy [13]. In addition, AA patients had increased HD biomarker cardiac troponin I in a previous report [14]. However, no differences were observed in cardiovascular risk between AA patients and controls in a previous cohort study [8]. Recently, a meta-analysis conducted by Amamoto et al. [20] reported that AA was not related to HDs (relative risk: 0.91; 95% CI: 0.60–1.39; p = 0.66). Also, Lee et al. [21] reported similar findings in their meta-analysis. Likewise, there were no consistent findings about the risk of HDs in cohort studies. In addition, the results of all these studies were statistically insignificant. This implies that patients with AA do not have a significantly higher or lower risk of associated HDs.

There was no dose-dependent relationship among AA severity, duration, and incidence of HDs in the present study. Even, there were no cases of HDs in the severe AA group. In addition, the adjusted HR was 0.89 for long-standing AA patients, while it was 1.22 for patients with episodic AA.

Increasing evidence suggests that AA does not extend to systemic inflammation. Psoriasis, which is known to have a significantly higher risk of associated HDs [22], is associated with higher C-reactive protein (CRP) levels. Moreover, serum CRP level is one of the factors indicating the severity of psoriasis [23,24]. However, a recent study reported that levels of serum homocysteine or high-sensitivity CRP were not increased in AA patients [25]. And studies evaluating the risk of HDs in AA patients have not shown consistent results so far. A study by Huang et al. found that AA was not related to an increased risk of acute myocardial infarction without statistical significance [8]. In addition, Lee et al. showed the risk of cardiovascular mortality is not associated with presence of AA [26]. However, Shin et al. claimed significantly increased risk of acute myocardial infarction in patients with AA only in the later stage of the observation [27].

Our results must be interpreted in the context of our study design. Diagnostic data were obtained from a national healthcare database, which could lead to errors. There could be a possibility that enrolled patients had AA or HD before they received diagnostic codes from the database. In order to resolve this problem, we set the first two years as a washout period. Patients who received the AA diagnostic codes during this period were not enrolled in our study. There could be significant differences in demographic characteristics between the AA group and the control group while selecting each member. Therefore, we adjusted for such confounding factors while analyzing the hazard ratios. In conclusion, the results of this large retrospective cohort study suggested that patients with AA did not show an increased risk for HDs.

24 Feb 2021

PONE-D-21-00164

Alopecia areata is not a risk factor for heart diseases: A 10-year retrospective cohort study

PLOS ONE

Dear Dr. Choi,

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.

Please submit your revised manuscript by Apr 04 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

We look forward to receiving your revised manuscript.

Kind regards,

Feroze Kaliyadan, M.D.

Academic Editor

PLOS ONE

Journal Requirements:

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. Please ensure that you include a title page within your main document. We do appreciate that you have a title page document uploaded as a separate file, however, as per our author guidelines (http://journals.plos.org/plosone/s/submission-guidelines#loc-title-page) we do require this to be part of the manuscript file itself and not uploaded separately.

Could you therefore please include the title page into the beginning of your manuscript file itself, listing all authors and affiliations.

3. Thank you for providing the date(s) when patient medical information was initially recorded. Please also include the date(s) on which your research team accessed the databases/records to obtain the retrospective data used in your study.

4. Thank you for stating in the text of your manuscript "The present study used de-identified data and was approved by the Institutional Review Board of the Ajou University Hospital (IRB-No: AJIRB-MED-EXP-18-404)". Please also add this information to your ethics statement in the online submission form.

5. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer #1: The study examined the data of a large cohort of Asian patients diagnosed with alopecia areata with regard to their risk of developing heart diseases. A few clarifications:

Were smoking/ substance abuse assessed as potential confounders. If potential confounders have been missed, add them as limitations.

The increased number of dyslipidemic patients in the AA group- whether on treatment, potential implications.

The authors may discuss the mechanistics of AA and heart disease, a little more in depth (some authors have hinted at a benefit, as well).

Reviewer #2: In this observational epidemiological study, the authors investigate a possible association between Alopecia Areata (AA) and Heart Disease (HD). They use a prospective cohort with a regression model to understand this relationship. They were able to use a national insurance sample to compare almost 4000 cases with 18850 matched cohorts. No association was found in univariate or multivariate analysis between AA and HD. The paper has the advantages of being a rigorous epidemiological study that does accomplish what it set out to do. The authors were able to find a large cohort and matched samples. Their statistical analysis is credible and their findings are consistent with the available literature in this field. The problem lies in the premise of the study as there is only a tenuous association between AA and HD in the current literature, questioning the need for such an endeavor. The authors also try to understand if there is a relationship between severe AA and HD and there appears to be no basis for considering this. Overall, this study adds to the slim body of evidence that AA does not appear to have any correlation with incidence of HD

Major Issues

Unclear as to how ethnic differences would affect any potential association between AA and HD. The authors briefly mention this but do not elaborate.

Minor Issues

1. Arbitrary definition of long standing AA as greater than one year

2. Authors do not clarify why they chose a 1:5 control ratio and if a smaller (or larger) ratio would have had an impact on the findings

3. Dyslipidemia was more common in AA group but it would have biased the results in favor of an association. Hence, this is likely not relevant

4. ICD code used for heart failure needs to be identified

5. Chronic myocardial infarction is a poorly defined entity. I suspect the authors were referencing ICD I25.2 (old myocardial infarction)

Reviewer #3: This large retrospective cohort study explores the relationship between AA as a chronic inflammatory disorder (like psoriasis, atopic dermatitis, vitiligo…) and heart disease in 3770 patients. Even the subgroups of severe AA (Ophiasis, totalis, universalis) and long standing AA (more than a year per disease duration) failed to show a statistically significant causal association with heart disease (Heart failure, myocardial infarction, angina) and comorbidities over a 9 year study period. I congratulate the author on a well conducted original research paper.

**********

[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.

26 Mar 2021

Dear Editor,

Thank you for your decision regarding our manuscript.

After we have carefully considered the reviewer's comments, we have revised the manuscript accordingly. We hope that you find this revised version acceptable for publication in your distinguished journal.

Thank you for your valuable comments.

----------------------------------------------------------------------------------

Reviewer comments:

Reviewer 1 #1: A few clarifications: Were smoking/ substance abuse assessed as potential confounders. If potential confounders have been missed, add them as limitations.

Authors’ response:

- As the reviewer pointed out, smoking could affect the results. Therefore, we analyzed smoking related diseases, such as stress disorders, peripheral vascular diseases, atherosclerosis, and stroke, which could more directly affect heart diseases than smoking.

- Also, in Korea, substance abuse is very rare that we did not need to take consideration. Also, due to its rarity, we could not reach such information by Korean National Health Insurance Service-National Sample Cohort.

Reviewer 1 #2: The increased number of dyslipidemic patients in the AA group- whether on treatment, potential implications.

Authors’ response:

- As the reviewer pointed out, whether to be treated dyslipidemia could act as a bias. Thus, we adjusted such confounding factor ‘the presence of dyslipidemia’ when seeking hazard ratios, that we could be free from the possibility of the bias.

Reviewer 1 #3: The authors may discuss the mechanistics of AA and heart disease, a little more in depth (some authors have hinted at a benefit, as well).

Authors’ response:

- So far, the detailed mechanisms of alopecia areata are unclear. However, we could have been postulated that AA could be related to heart diseases by the various findings (Wang E.H., et al. Alopecia Areata is Associated with Increased Expression of Heart Disease Biomarker Cardiac Troponin I. Acta Derm Venereol. 2018;98(8):776-82; Wang E. et al. Development of autoimmune hair loss disease alopecia areata is associated with cardiac dysfunction in C3H/HeJ mice. PLoS One. 2013;8(4):e62935), and conducted this study. To deliver such perspective more clearly, we added this concept in the INTRODUCTION (Line 56-58)

Reviewer 2 #1: Unclear as to how ethnic differences would affect any potential association between AA and HD. The authors briefly mention this but do not elaborate.

Authors’ response:

- As we can see in other studies (Lane et al, Ethinic differences in blood pressure and the prevalence of hypertension in England. Journal of Human Hypertension 2002; 16: 267–273; Rampal et al, Ethinic differences in the prevalence of metabolic syndrome: Results from a multi-ethinic population-based survey in Malaysia, Plos One, 2012; 7(9): e46365), prevalence of various metabolic disease differs by the ethnicity. Because of the cultural and genetic differences, vulnerability to such diseases which are largely related to heart diseases could be varied by ethnicity.

- Thus, we were about to confirm the irrelevance between AA and cardiovascular disease, further general heart diseases by the large population of the Korean.

- To deliver our intention clearly, we inserted such contents in the INTRODUCTION. (line 61-62)

Reviewer 2 #2: Arbitrary definition of long-standing AA as greater than one year.

Authors’ response:

- As approximately 60% of AA patients have at least a partial hair regrowth by 1 year (according to Kang et al. Fitzpatrick’s Dermatology, 9th edi.), previous studies (McDonald Hull et al. Guidelines for the management of alopecia areata. British J Dermatol 2003;149: 692–699; Pratt et al. Alopecia areata. Nat Rev Dis Primers. 2017; 3:17011) have used such criteria for ‘long-standing AA’. Thus, we defined ‘long-standing AA’ as more than a year, as well.

Reviewer 2 #3: Authors do not clarify why they chose a 1:5 control ratio and if a smaller (or larger) ratio would have had an impact on the findings.

Authors’ response:

- According to Hennessy et al. statistical power can be achieved when more than five controls per case were analyzed in case-control study design. (Hennessy et al. Factors Influencing the Optimal Control-to-Case Ratio in Matched Case-Control Studies. American Journal of Epid 1999; 149(2))

Reviewer 2 #4: Dyslipidemia was more common in AA group but it would have biased the results in favor of an association. Hence, this is likely not relevant.

Authors’ response:

- As the reviewer pointed out, whether to be treated dyslipidemia could act as a bias. Thus, we adjusted such confounding factor ‘the presence of dyslipidemia’ when seeking hazard ratios, that we could be free from the possibility of the bias.

Reviewer 2 #5: ICD code used for heart failure needs to be identified.

Authors’ response:

- Following this comment, we elaborated the ICD codes for heart diseases in METHODS (line 86-88).

Reviewer 2 #6: Chronic myocardial infarction is a poorly defined entity. I suspect the authors were referencing ICD I25.2 (old myocardial infarction).

Authors’ response:

- According to ICD-10 version of 2019, I25 code (chronic ischaemic heart disease) consists of I25.0 (Atherosclerotic cardiovascular disease, so described), I25.1 (Atherosclerotic heart disease), I25.2 (Old myocardial infarction), I25.3 (Aneurysm of heart), I25.4 (Coronary artery aneurysm and dissection), I25.5 (Ischaemic cardiomyopathy), I25.6 (Silent myocardial ischaemia), I25.8 (Other forms of chronic ischaemic heart disease), and I25.9 (Chronic ischaemic heart disease, unspecified)

- As those disease entities could be both a cause and effect of chronic myocardial infarction, we wanted to include all the subclassification codes in this study. To clarify this intention, we added the ICD-10 code description in METHODS. (line 86-88)

Submitted filename: Reponse to Reviewers.doc

Click here for additional data file.

5 Apr 2021

Alopecia areata is not a risk factor for heart diseases: A 10-year retrospective cohort study

PONE-D-21-00164R1

Dear Dr. Choi,

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,

Feroze Kaliyadan, M.D.

Academic Editor

PLOS ONE

27 Apr 2021

PONE-D-21-00164R1

Alopecia areata is not a risk factor for heart diseases: A 10-year retrospective cohort study

Dear Dr. Choi:

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. Feroze Kaliyadan

Academic Editor

PLOS ONE