# **Supplemental Online Content**

Rayinda T, McSweeney SM, Christou E, et al. Gene-environment interaction between *CYP1B1* and oral contraception on frontal fibrosing alopecia. *JAMA Dermatol*. Published May 29, 2024. doi:10.1001/jamadermatol.2024.1315

**eTable 1.** Summary of Risk Allele in *CYP1B1*

**eTable 2.** Result of Interaction Test Between Risk Allele rs1800440 (A) and Oral Contraceptive Pills (OCP) Using OCP Status and 4 First Principal Component Analysis as Covariates

**eFigure 1.** Case and Control Matching Based on Age and First 4 Principal Components (PC) From PC Analysis

**eFigure 2.** Principal Component Analysis After Case-Control Matching Based on the First 4 Principal Components (Based on 17 821 Variants and 34 774 People)

**eReference.** Reference for eFigure 2

**eMethods.** Study Design

**eReferences.** References for eMethods

This supplemental material has been provided by the authors to give readers additional information about their work.

# **eTable 1.** Summary of Risk Allele in *CYP1B1*

| Chromosome | 2 |
|-------------------------------------------|------------------------|
| Base position (hg19) | 38,298,139 |
| rsID | rs1800440 |
| Gene | CYP1B1 |
| Reference allele | A |
| Alternative allele | G |
| Protein change | c.1358A>G, p.Asn453Ser |
| Risk allele (A) frequency, based on dbSNP | Global: 0.829 |
| (https://www.ncbi.nlm.nih.gov/snp/)1 | European: 0.818 |

**eTable 2.** Result of Interaction Test Between Risk Allele rs1800440 (A) and Oral Contraceptive Pills (OCP) Using OCP Status and 4 First Principal Component Analysis as Covariates

| Test | OR | 95% CI | P-value |
|-----------------|---------|----------------------------------|-----------------|
| rs1800440 | 1.17 | 0.82 - 1.65 | 0.37 |
| OCP | 0.13 | 0.06 - 0.27 | -8 9.16 × 10 |
| Covariate 1 | 4 × 103 | 2.25 × 10-4 10 – 7.08 × 10 | 0.33 |
| Covariate 2 | 0.14 | 7.22 × 10-9 6 – 3.04 × 10 | 0.82 |
| Covariate 3 | 3.29 | 1.74 × 10-7 7 – 6.22 × 10 | 0.89 |
| Covariate 4 | 5.98 | 3.06 × 10-7 8 – 1.17 × 10 | 0.83 |
| rs1800440 × OCP | 1.63 | 1.07 - 2.46 | 0.02 |

**NOTE.** rs1800440× OCP, interaction test between rs1800440 under additive model and oral contraceptive pills status, CI, confident interval, OCP, oral contraceptive pills status, OR, odds ratio

**eFigure 1.** Case and Control Matching Based on Age and First 4 Principal Components (PC) From PC Analysis

![](https://assets.reachrx.ai/images/10.1001%E2%98%8Djamadermatol.2024.1315/_page_3_Figure_1.jpeg)

**eFigure 2.** Principal Component Analysis After Case-Control Matching Based on the First 4 Principal Components (Based on 17 821 Variants and 34 774 People)

![](https://assets.reachrx.ai/images/10.1001%E2%98%8Djamadermatol.2024.1315/_page_4_Figure_1.jpeg)

FFA cases are indicated by pink color.

**eReference.** Reference for eFigure 2

1. Sherry ST, Ward M, Sirotkin K. dbSNP-database for single nucleotide polymorphisms and other classes of minor genetic variation. *Genome Res*. 1999;9(8):677-679.

### **eMethods.** Study Design

# **Case and control populations**

Cases were defined as (1) women with FFA diagnosed by a consultant dermatologist and supported by histopathology, if necessary; (2) recruited to the UK FFA GWAS cohort1 between 2015 and 2017; (3) of white British ancestry; and (4) a history of taking a long-term oral contraceptive. Information regarding the duration and type of OCP usage was retrospectively collected from clinical case report forms during the sample collection process. Clinical and genotype data were initially available for 519 cases.

Control populations were selected from the UK-Biobank (approved project number 15147) according to the following criteria: (1) female individuals (data field 31) without a history of lichenoid alopecia (UK Biobank data field 131784), (2) individuals of European white ancestry (data field 21000), (3) individuals of UK origin (UK Biobank data field 1647), and (4) data available at baseline visit describing history of oral contraceptive use (data field 2784\_0\_0). Controls were excluded if there is any inconsistency in the follow up visits (data field f2784\_2\_1, f2784\_2\_2, and f2784\_2\_3). Initially, 244,987 controls data were available.

## **Genotyping and genotype data quality control**

Genotyping of the FFA cases was performed using the Infinium OmniExpressExome BeadChip array from Illumina. For UK Biobank participants, genotypes were generated by Affymetrix using either the BiLEVE Axiom Array or the UK Biobank Axiom Array from Applied Biosystems in Waltham, MA. The initial quality control procedures were independently undertaken by the UK Biobank analysis team.2

Afterwards, we retained variants with call rates exceeding 99% in both the cases and controls, and those that did not deviate from the Hardy-Weinberg Equilibrium (P > 10−4). Additionally, we excluded individuals with a call rate less than 99% or extensive heterozygosity. Using a subset of 17,821 variants in linkage equilibrium (where the pairwise correlation coefficient r2 was less than 0.2), we evaluated the relatedness between individuals using the KING software package (version 2.2.5). We determined that no two individuals had estimated relatedness closer than the third

© 2024 American Medical Association. All rights reserved.

degree (Kinship coefficient >0.0442). Furthermore, a principal component analysis was performed on the same set of 17,821 variants, and the first four PCs were employed for further case-control matching.

#### **Case-control matching**

Cases and controls were matched based on the age, sex, and the status of OCP use, using the R software package MatchIt (http://cran.r- project.org/we b/packages/MatchIt/vignett es/MatchIt.html). The "nearest" method (which implements greedy nearest neighbor matching) was selected to select control samples that most closely matched the FFA cases using propensity scores, and adding controls until the standardized mean difference between cases and controls reached the recommended upper limit of 0.1, resulting in a case:control ratio of 1:66.

## **Genetic model**

Before commencing association and interaction testing in the matched FFA-UKB dataset, we confirmed using the full UK and Spanish genotype data from our 2019 meta-analysis1 that an additive genetic model is preferable to other models (autosomal recessive, autosomal dominant) to describe the observed association between rs1800440 genotype and FFA (results not shown). All subsequent analysis therefore assumes an additive model.

## **Association test of rs1800440 and FFA**

The association between the risk allele in *CYP1B1* at 2p22.2 (rs1800440) and FFA was undertaken in PLINK 1.9 using an additive logistic regression model. Principal components were incorporated as additional covariates to control for residual population structure. The number of covariates from principal components was chosen based on inspection of the scree plot (n=4).

## **Gene-environment interaction tests**

The interaction between the risk allele in *CYP1B1* at 2p22.2 (rs1800440) and OCP status as a covariate was evaluated in PLINK 1.9 using a logistic regression model with an interaction term, under an additive genetic model. Principal components were incorporated as additional covariates to control for residual population structure. The number of covariates from principal components was chosen based on inspection of the scree plot (n=4).

# **eReferences.** References for eMethods

- 1. Tziotzios C, Petridis C, Dand N, et al. Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B\*07:02. *Nat Commun*. 2019;10(1):1150. doi:10.1038/s41467-019-09117-w.
- 2. Bycroft C, Freeman C, Petkova D, et al. The UK Biobank resource with deep phenotyping and genomic data. *Nature*. 2018;562(7726):203-209. doi:10.1038/s41586-018-0579-z.

JAMA Dermatology

<jats:sec id="ab-dbr240005-4"><jats:title>Importance</jats:title><jats:p>Frontal fibrosing alopecia (FFA) is an increasingly prevalent form of follicular lichen planus, causing irreversible hair loss predominantly in postmenopausal individuals. An earlier genome-wide meta-analysis of female FFA identified risk loci in genes implicated in self-antigen presentation and T-cell homeostasis, including HLA-B*07:02, <jats:italic>ST3GAL1</jats:italic>, and <jats:italic>SEMA4B</jats:italic>. However, <jats:italic>CYP1B1</jats:italic>, which is important for hormone metabolism, was also implicated with the substitution of serine for asparagine at position 453 (c.1358A&amp;amp;gt;G, p.Asn453Ser) exhibiting a protective effect against FFA. Increasing understanding of genetic and environmental variables and their interactions will improve understanding of disease pathogenesis and has the potential to inform risk mitigation strategies.</jats:p></jats:sec><jats:sec id="ab-dbr240005-5"><jats:title>Objective</jats:title><jats:p>To investigate whether oral contraceptive pill (OCP) use modulates the protective effect of the common missense variant in <jats:italic>CYP1B1</jats:italic> (c.1358A&amp;amp;gt;G, p.Asn453Ser) on FFA risk.</jats:p></jats:sec><jats:sec id="ab-dbr240005-6"><jats:title>Design, Setting, and Participants</jats:title><jats:p>This gene-environment interaction study using a case-control design enrolled female patients with FFA from UK-based dermatology clinics. The patients were matched with unrelated age- and ancestry-matched female control individuals derived from UK Biobank in a 1:66 ratio, determined by the first 4 principal components from genome-wide genotypes. Data were collected from July 2015 to September 2017, and analyzed from October 2022 to December 2023.</jats:p></jats:sec><jats:sec id="ab-dbr240005-7"><jats:title>Main Outcome and Measure</jats:title><jats:p>The main outcomes were the modulatory effect of OCP use on the contribution of the <jats:italic>CYP1B1</jats:italic> missense variant to female FFA risk and a formal gene-environment interaction test evaluated by a logistic regression model with a multiplicative interaction term, under the assumptions of an additive genetic model interaction term, under the assumptions of an additive genetic model.</jats:p></jats:sec><jats:sec id="ab-dbr240005-8"><jats:title>Results</jats:title><jats:p>Of the 489 female patients with FFA, the mean (SD) age was 65.8 (9.7) years, and 370 (75.7%) had a history of OCP use. Of the 34 254 age- and ancestry-matched control individuals, the mean (SD) age was 65.0 (8.4) years, and previous OCP use was reported in 31 177 (91.0%). An association between female FFA and the <jats:italic>CYP1B1</jats:italic> risk allele was observed in individuals who reported OCP use (odds ratio, 1.90 [95% CI, 1.50-2.40]; <jats:italic>P</jats:italic> = 8.41 × 10<jats:sup>−8</jats:sup>) but not in those with no documented exposure to OCPs (odds ratio, 1.16 [95% CI, 0.82-1.64]; <jats:italic>P</jats:italic> = .39). A full gene-environment interaction model demonstrated a significant additive statistical interaction between c.1358A, p.453Asn, and history of OCP use on FFA risk (OR for interaction, 1.63 [95% CI, 1.07-2.46]; P = .02)</jats:p></jats:sec><jats:sec id="ab-dbr240005-9"><jats:title>Conclusions and Relevance</jats:title><jats:p>This gene-environment interaction analysis suggests that the protective effect of the <jats:italic>CYP1B1</jats:italic> missense variant on FFA risk might be mediated by exposure to OCPs. The allele that encodes an asparagine at position 453 of <jats:italic>CYP1B1</jats:italic> was associated with increased odds of FFA only in participants with OCP history.</jats:p></jats:sec>

Cytochrome P450 1B1: role in health and disease and effect of nutrition on its expression.

RSC Adv

Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B*07:02.

Nat Commun

Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.

Mol Pharmacol

Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by benzo(a)pyrene ex vivo.

Mutagenesis

Catalytic properties of polymorphic human cytochrome P450 1B1 variants.

Carcinogenesis

Frontal fibrosing alopecia: an example of disrupted aryl hydrocarbon receptor–mediated immunological homeostasis in the skin?

Clin Cosmet Investig Dermatol

Frontal fibrosing alopecia: an update on pathogenesis, diagnosis, and treatment.

Am J Clin Dermatol

Risk factors associated with frontal fibrosing alopecia: a multicentre case-control study.

Clin Exp Dermatol

Frontal fibrosing alopecia: reflections and hypotheses on aetiology and pathogenesis.

Exp Dermatol

History of oral contraceptive drugs and their use worldwide.

Best Pract Res Clin Endocrinol Metab

Shared genetic risk variants in both male and female frontal fibrosing alopecia.

J Invest Dermatol

Comparison of sociodemographic and health-related characteristics of UK Biobank participants with those of the general population.

Am J Epidemiol

Patterns of oral contraceptive use in the United Kingdom.

Br J Rheumatol

Frontal fibrosing alopecia (FFA) is an increasingly prevalent form of follicular lichen planus, causing irreversible hair loss predominantly in postmenopausal individuals. An earlier genome-wide meta-analysis of female FFA identified risk loci in genes implicated in self-antigen presentation and T-cell homeostasis, including HLA-B*07:02, ST3GAL1, and SEMA4B. However, CYP1B1, which is important for hormone metabolism, was also implicated with the substitution of serine for asparagine at position 453 (c.1358A>G, p.Asn453Ser) exhibiting a protective effect against FFA. Increasing understanding of genetic and environmental variables and their interactions will improve understanding of disease pathogenesis and has the potential to inform risk mitigation strategies.
To investigate whether oral contraceptive pill (OCP) use modulates the protective effect of the common missense variant in CYP1B1 (c.1358A>G, p.Asn453Ser) on FFA risk.
This gene-environment interaction study using a case-control design enrolled female patients with FFA from UK-based dermatology clinics. The patients were matched with unrelated age- and ancestry-matched female control individuals derived from UK Biobank in a 1:66 ratio, determined by the first 4 principal components from genome-wide genotypes. Data were collected from July 2015 to September 2017, and analyzed from October 2022 to December 2023.
The main outcomes were the modulatory effect of OCP use on the contribution of the CYP1B1 missense variant to female FFA risk and a formal gene-environment interaction test evaluated by a logistic regression model with a multiplicative interaction term, under the assumptions of an additive genetic model interaction term, under the assumptions of an additive genetic model.
Of the 489 female patients with FFA, the mean (SD) age was 65.8 (9.7) years, and 370 (75.7%) had a history of OCP use. Of the 34 254 age- and ancestry-matched control individuals, the mean (SD) age was 65.0 (8.4) years, and previous OCP use was reported in 31 177 (91.0%). An association between female FFA and the CYP1B1 risk allele was observed in individuals who reported OCP use (odds ratio, 1.90 [95% CI, 1.50-2.40]; P = 8.41 × 10-8) but not in those with no documented exposure to OCPs (odds ratio, 1.16 [95% CI, 0.82-1.64]; P = .39). A full gene-environment interaction model demonstrated a significant additive statistical interaction between c.1358A, p.453Asn, and history of OCP use on FFA risk (OR for interaction, 1.63 [95% CI, 1.07-2.46]; P = .02).
This gene-environment interaction analysis suggests that the protective effect of the CYP1B1 missense variant on FFA risk might be mediated by exposure to OCPs. The allele that encodes an asparagine at position 453 of CYP1B1 was associated with increased odds of FFA only in participants with OCP history.

Gene-Environment Interaction Between CYP1B1 and Oral Contraception on Frontal Fibrosing Alopecia.

Gene-Environment Interaction Between CYP1B1 and Oral Contraception on Frontal Fibrosing Alopecia

Gene-Environment Interaction Between <i>CYP1B1</i> and Oral Contraception on Frontal Fibrosing Alopecia

Clinical Summary

What was studied

Key findings

Study limitations

Related Questions