# Genome-Wide Association Study of Fluorescent Oxidation Products Accounting for Tobacco Smoking Status in Adults from the French EGEA Study

Laurent Orsi, Patricia Margaritte-Jeannin, Miora Andrianjafimasy, Orianne Dumas, Hamida Mohamdi, Emmanuelle Bouzigon, Florence Demenais, Régis Matran, Farid Zerimech, Rachel Nadif, et al.

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Laurent Orsi, Patricia Margaritte-Jeannin, Miora Andrianjafimasy, Orianne Dumas, Hamida Mohamdi, et al.. Genome-Wide Association Study of Fluorescent Oxidation Products Accounting for Tobacco Smoking Status in Adults from the French EGEA Study. Antioxidants, 2022, 11 (5), pp.802. 10.3390/antiox11050802. [https://doi.org/10.3390/antiox11050802](https://doi.org/10.3390/antiox11050802)

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Article

# Genome-Wide Association Study of Fluorescent Oxidation Products Accounting for Tobacco Smoking Status in Adults from the French EGEA Study

Laurent Orsi<fnref n="1" />,*, Patricia Margaritte-Jeannin<fnref n="2" />,†, Miora Andrianjafimasy <fnref n="1" />,†, Orianne Dumas<fnref n="1" />,†, Hamida Mohamdi<fnref n="2" />, Emmanuelle Bouzigon<fnref n="2" />, Florence Demenais<fnref n="2" />, Régis Matran<fnref n="3" />,<fnref n="4" />,<fnref n="5" />, Farid Zerimech<fnref n="3" />,<fnref n="4" />,<fnref n="5" />,‡, Rachel Nadif <fnref n="1" />,‡ and Marie-Hélène Dizier <fnref n="2" />,‡

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**Citation:** Orsi, L.; Margaritte-Jeannin, P.; Andrianjafimasy, M.; Dumas, O.; Mohamdi, H.; Bouzigon, E.; Demenais, F.; Matran, R.; Zerimech, F.; Nadif, R.; et al. Genome-Wide Association Study of Fluorescent Oxidation Products Accounting for Tobacco Smoking Status in Adults from the French EGEA Study.
*Antioxidants* **2022**, *11*, 802. https://doi.org/10.3390/antiox11050802

Academic Editor: Stephen M. Black

Received: 17 February 2022

Accepted: 18 April 2022

Published: 20 April 2022

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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Abstract:** Oxidative stress (OS) is the main pathophysiological mechanism involved in several chronic diseases, including asthma. Fluorescent oxidation products (FlOPs), a global biomarker of damage due to OS, is of growing interest in epidemiological studies. We conducted a genome-wide association study (GWAS) of the FlOPs level in 1216 adults from the case-control and family-based EGEA study (mean age 43 years old, 51% women, and 23% current smokers) to identify genetic variants associated with FlOPs. The GWAS was first conducted in the whole sample and then stratified according to smoking status, the main exogenous source of reactive oxygen species. Among the top genetic variants identified by the three GWAS, those located in *BMP6* (*p* = 3 × 10⁻⁶), near *BMPER* (*p* = 9 × 10⁻⁶), in *GABRG3* (*p* = 4 × 10⁻⁷), and near *ATG5* (*p* = 2 × 10⁻⁹) are the most relevant because of both their link to biological pathways related to OS and their association with several chronic diseases for which the role of OS in their pathophysiology has been pointed out. *BMP6* and *BMPER* are of particular interest due to their involvement in the same biological pathways related to OS and their functional interaction. To conclude, this study, which is the first GWAS of FlOPs, provides new insights into the pathophysiology of chronic OS-related diseases.

**Keywords:** fluorescent oxidation products; oxidative stress; genome-wide association study; chronic diseases; asthma; smoking

## 1. Introduction

Oxidative stress (OS) was defined in 1985 as "a disturbance in the pro-oxidant/antioxidant balance in favour of the former" [1]. Beyond its essential role in life processes, OS is involved in the pathophysiology of several chronic diseases, including cardiovascular diseases, chronic kidney diseases, and asthma [2]. Sources of reactive oxygen species include the diseases themselves, through their intracellular metabolisms, and some exogenous sources, among which the most important is cigarette smoke [3].

Among the numerous biomarkers related to OS [1,4], Fluorescent Oxidation Products (FlOPs), which reflect a global measurement of oxidation of lipids, proteins, carbohydrates,

<footnote n="1">¹ Université Paris-Saclay, UVSQ, Univ. Paris-Sud, INSERM, Équipe d'Epidémiologie Respiratoire Intégrative, CESP, 94807 Villejuif, France; mioraandria3@gmail.com (M.A.); orianne.dumas@inserm.fr (O.D.); rachel.nadif@inserm.fr (R.N.)</footnote>

<footnote n="2">² Université Paris Cité, INSERM, UMR 1124, Group of Genomic Epidemiology and Multifactorial Diseases, 75006 Paris, France; patricia.jeannin@inserm.fr (P.M.-J.); hamida.mohamdi@inserm.fr (H.M.); emmanuelle.bouzigon@inserm.fr (E.B.); florence.demenais@inserm.fr (F.D.); marie-helene.dizier@inserm.fr (M.-H.D.)</footnote>

<footnote n="3">³ Univ. Lille, ULR 4483—IMPECS, 59000 Lille, France; regis.matran@univ-lille.fr (R.M.); farid.zerimech@chu-lille.fr (F.Z.)</footnote>

<footnote n="4">⁴ CHU Lille, 59000 Lille, France</footnote>

<footnote n="5">⁵ Institut Pasteur de Lille, 59000 Lille, France</footnote>

<footnote>* Correspondence: laurent.orsi@inserm.fr</footnote>

<footnote>† These authors contributed equally to the work.</footnote>

<footnote>‡ These authors contributed equally to the work.</footnote>
and DNA [5], is of growing interest for epidemiological studies [6], as an easily quantifiable
and stable biomarker of damage due to OS. The FIOPs level was found to be associated with
chronic diseases such as coronary heart disease (CHD) (e.g., incidence of CHD among men
without previous cardiovascular events, and risk of future CHD in healthy women) [7,8],
and chronic kidney diseases [9]. Recently, we reported that high FIOPs level was associated
with asthma attacks, the use of any asthma treatment, and poor control of asthma and
was a predictor of asthma evolution in adults from the French Epidemiological study on
the Genetics and Environment of Asthma (EGEA) [10,11]. We also found that smoking
increased the FIOPs level [11].

Understanding the aetiology of multifactorial and heterogeneous chronic diseases is challenging. We hypothesised that genome-wide association studies (GWAS) of FIOPs may provide new insights into the pathophysiology of complex chronic diseases related to the OS pathway, including asthma. Indeed, previous GWAS of levels of circulating protein biomarkers related to chronic obstructive pulmonary disease, another chronic lung disease, was useful to identify new genes linked to this disease [12]. Such an approach was also useful to identify new genes through the study of biomarkers of other chronic diseases such as kidney or cardiovascular diseases [13,14]. To our knowledge, no GWAS of biomarkers related to the OS pathway, and in particular FIOPs, has been published to date.

Taking advantage of the extensive biological, genotypic, and phenotypic characteri-
sation of >1000 adults from the EGEA study, we aimed to identify genetic loci associated
with the FIOPs level through a GWAS. As smoking is the main environmental source of OS
and is associated with the FIOPs level, we performed two supplementary GWAS analyses
separately in contrasted groups according to tobacco smoking status that could help us to
identify genetic loci that could have been missed in the whole sample.

**2. Materials and Methods**

2.1. *Study Population (EGEA Study)*

EGEA is a French cohort study based on an initial group of asthma cases recruited in five chest clinics from Grenoble, Lyon, Marseille, Montpellier, and Paris, along with their first-degree relatives, and population-based controls (EGEA1, 1991–1995). The protocol of the study has been described previously [15,16]. Briefly, the asthma cases and their first-degree relatives were recruited from respiratory or allergic clinics. The adult cases were recruited in the five cities, and the child cases were recruited in Paris, Grenoble, and Marseille. Control adults were recruited from electoral rolls in Paris, Lyon, Montpellier, and Grenoble, a check-up centre in Marseille, and surgery clinic from the same hospital in Paris and Grenoble. Control children were always recruited from surgery clinics. Overall matching by month of exam, age decade, sex, and centre was done. A first follow-up of the initial cohort was conducted between 2003 and 2007 (EGEA2) including 1602 participants (98% adults) with complete examination. At each survey, all participants answered standardised and validated questionnaires to identify asthma and to determine respiratory and allergic symptoms, treatments, environmental exposures, and lifestyle characteristics, including tobacco smoking status. More details are given in Supplementary Materials. The data used for the present analyses were elicited at EGEA2.

The EGEA collection was certified ISO 9001 from 2006 to 2018 [17]. All participants signed a written informed consent, and ethical approval was obtained from the relevant institutional review board committees (Cochin Port-Royal Hospital and Necker-Enfants Malades Hospital, Paris, France).

2.2. FIOPs Level

Plasma samples were collected in EGEA2 between 2003 and 2006 and stored immedi-
ately at -80 °C during 5.0 to 8.0 years until FIOPs measurements. The plasma FIOPs level
was measured as previously described [7]. Briefly, plasma was extracted into a mixture
of ethanol/ether (3/1 v/v) and measured using a spectrofluorometer (360 nm excitation
wavelength, 430 nm emission wavelength). Fluorescence was expressed as a unit of relative
fluorescence intensity (RFU)·mL⁻¹ of plasma. Each sample was replicated. The intra-assay coefficient of variation (CV) for FIOPs was less than 20%. The dosages for which the CV were ≥20 % or those that were haemolysed were removed of analysis (n = 11 and n = 8, respectively, see Figure S1 in Supplementary Materials).

## 2.3. Genotyping

The EGEA participants were genotyped using Illumina 610 Quad array at the Centre National de Génotypage (CNG, Evry, France) as part of the European Gabriel consortium asthma GWAS [18]. As part of this consortium, principal-components analysis was conducted for all participants to control population admixture and was carried out using the EIGENSTRAT2.0 software. Putative non-European samples were flagged as outliers and eliminated from subsequent analyses [18]. Stringent quality control (QC) criteria were used to select both individuals and genotyped Single Nucleotide Polymorphisms (SNPs) for analysis [19]. Among participants with genotyped data (n = 1481), 44 with invalid genotyped data were excluded (see Supplementary Materials: Figure S1, flow chart, for details). The following SNPs quality controls were applied: genotyping call rates ≥ 97% and departure from the Hardy-Weinberg equilibrium in the controls (p-value ≥ 1.0 × 10⁻⁴) and minor allele frequencies (MAF) ≥ 5%. After this SNPs QC control, 66,422 SNPs were excluded, and a total of 501 167 SNPs were available for the analysis. To investigate regions of interest, including the top three SNPs, imputed SNPs from the reference panel 1000 Genome Phase I were used [20]. The software IMPUTE2 was used for imputation [21]. Imputed SNPs were kept for analysis if their imputation information score was greater than or equal to 0.70 and if their minor allele frequency (MAF) was greater than or equal to 0.05.

## 2.4. Definitions of Population's Characteristics

### 2.4.1. Asthma

Ever-asthma status was generated as a dichotomous variable (never-asthmatic/ever-asthmatic). Ever-asthmatics were participants who answered positively to at least one of the two following questions: "Have you ever had attacks of breathlessness at rest with wheezing?" or "Have you ever had asthma attacks?", or those who were recruited as asthmatic cases at EGEA1. Never-asthmatics were those who answered negatively to the two questions above; they were not recruited as asthmatic cases at EGEA1.

### 2.4.2. Chronic Bronchitis

Chronic bronchitis was generated as a dichotomous variable (yes/no). Participants with chronic bronchitis were those who answered positively to at least one of the two following questions: "Do you usually cough during the day or at night in winter almost every day for three months of continued every year?" or "Do you usually spit during the day or at night in winter, almost every day for three months of continued every year?".

### 2.4.3. Lung Function

A lung function test with spirometry and methacholine challenge was performed using standardised protocol with similar equipment across centres and according to the American Thoracic Society/European Respiratory Society guidelines [22]. The forced expiratory volume in one second (FEV₁) percent predicted value was based on Quanjer et al. reference equations [23]. For participants with a FEV₁ ≥ 80% of the predicted value, a methacholine bronchial challenge test was performed (maximum dose 4 mg) using a Biomedin spirometer (Biomedin Srl, Padua, Italy) in all centres, except in Lyon, where a Pneumotach Jaeger spirometer (Jaeger) was used. The following measures of lung function were used as continuous variables and expressed as %: FEV₁ and Forced Vital Capacity (FVC). FEV₁ was also generated as a dichotomous variable (FEV₁ < 80%, FEV₁ ≥ 80%).
#### 2.4.4. Smoking Status

Tobacco consumption was defined by the answer to the question "Do you smoke or have you smoked previously one cigarette per day or more, for at least a year?". If so, participants were asked their age at the start of smoking and the age of quitting, if applicable. Participants were also asked to quantify the average daily consumption of cigarettes and the average weekly consumption of cigars, if applicable. Current smoking status was generated as a 3-class categorical variable: never-smoker, ex-smoker, or current smoker. Lifelong cumulative quantity of tobacco was generated as a continuous variable and also categorised using a 4-class variable, with cut-offs defined a priori: never-smokers; <10 pack-year; 10–20 pack-year; and >20 pack-year.

#### 2.4.5. Body Mass Index (BMI)

BMI was generated as a continuous variable and was also expressed as a dichotomous variable (<30 kg/m²; ≥30 kg/m²).

#### 2.4.6. Biological Parameters

Total serum Immunoglobulin E (IgE) determination was assessed by the UniCAP system (Pharmacia®) from blood samples in a centralised laboratory and expressed in international units (IU) per millilitre. For the analysis, total IgE level was examined as a continuous variable.

Blood neutrophil and eosinophil counts were expressed in cells/mm³ and coded as continuous variables [24,25].

### 2.5. Statistical Methods and Strategy of Analysis

#### 2.5.1. Characteristics of the Studied Population and Association with the FLOPs Level

First, characteristics of the studied population were described and summarised as n (%) or mean (m) ± standard deviation (sd), according to the type of variable, either quantitative or qualitative. Due to their skewed distribution, the FLOPs level was log-transformed and expressed as geometric mean (GM) and values of first quartile (Q1) and third quartile (Q3).

In order to select potential confounding factors prior to GWAS, we estimated associations between log-FLOPs and several characteristics of the whole sample using Gaussian linear models, taking into account EGEA family structure, by Generalised Estimated Equation (GEE, SAS v9.4 (SAS Institute, Cary, NC, USA), proc genmod, option repeated). We previously identified the following characteristics as factors associated with FLOPs: age, sex, current smoking status, lifelong cumulative quantity of tobacco, blood neutrophil count, and FEV₁. Age was entered either as a continuous variable or a categorical one, with cut-points defined a priori (<25 years; 25–34 years; 35–44 years; 45–54 years; and ≥55 years). Regarding tobacco smoking, models included either smoking status as a categorical variable (never smokers; ex-smokers; and current smokers), or lifelong cumulative quantity of tobacco as a continuous variable or a 4-class categorical variable. The best model was selected based on the QIC, an Akaike's Information Criterion in the framework of GEE models [26]. Among all models tested, the best model included age (continuous), sex, and lifelong cumulative quantity of tobacco (never-smokers; <10 pack-year; 10–20 pack-year; and >20 pack-year).

Adjusted log-FLOPs were obtained as residuals of the best linear model identified in the previous step. Z-scores were then obtained by standardizing residuals, and adequacy to Gaussian distribution was assessed using the Kolmogorov test. In order to exclude participants whose log-FLOPs were poorly predicted by the linear model, participants with the highest Z-score (i.e., |Z-score| > 3, corresponding to the 0.1th and 99.9th percentiles of a standard Gaussian distribution) were excluded. The process was repeated until no significant deviation from Gaussian distribution was evidenced. This process excluded 20 participants (see Figure S1 in Supplementary Materials).
Adjusted log-FIOPs used for GWAS stratified on smoking status (see below) were gen-
erated by applying the same procedure as described above, except that lifelong cumulative
quantity of tobacco was not entered in the model.

2.5.2. GWAS of the FIOPs Level

We first conducted a GWAS of the FIOPs level in the whole sample with genotyped
data. Then, we conducted two supplementary GWAS separately in contrasted groups
according to smoking status at the time of measurement: never-smokers and current
smokers. Ex-smokers were excluded from this analysis.

An association analysis between adjusted log-FIOPs (standardised residual) and each SNP was performed by the Gaussian linear model, adjusted for principal components (PCs) to account for within European diversity. The EGEA family structure was taken into account using a robust variance estimator for clustered data (STATA command: regress, option vce.cluster), within family). SNPs were coded under an additive genetic model.

For the top three SNPs obtained by each of the three GWAS (in the whole sample, in never-smokers and in current smokers), further analyses were conducted. First, we split the sample into two actual independent sub-samples regarding the ascertainment mode (controls vs. cases/relatives) to check the consistency of the results by using a homogeneity test between the sub-samples. Second, due to the mode of ascertainment of EGEA families, i.e., through asthmatic participants, the independence of the results regarding the asthma status was verified by homogeneity test according to ever-asthma status (never-asthmatics vs. ever-asthmatics). A test for homogeneity was performed by fitting the interaction term between the SNP and dummy variables (cases/related vs. controls, and ever-asthmatics vs. never-asthmatics, respectively) in models. The top three SNPs of the three GWAS (in the whole sample, the never-smokers and the current smokers) was also focused on: we used imputed data from the reference panel 1000 Genome project Phase 1 [20] CEU population, spanning 500 kb on each side of each top SNP. For each region of interest, association results were graphically represented using LocusZoom [27].

All analyses were performed using SAS v9.4 (SAS Institute, Cary, NC, USA) or STATA v14.1 (StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX, USA: StataCorp LP). All tests were two-sided. To account for multiple testing, the Bonferroni-corrected significance *p*-value threshold applied to the Meff (effective number of independent tests after discarding dependence due to linkage disequilibrium (LD) between the SNPs) was calculated. For a chip of 610K SNPs, the significance *p*-value threshold was estimated to be 1.3 × 10<sup>-7</sup> [28].

2.5.3. eQTLs, meQTLs, and Functional Annotations

We investigated whether the top three SNPs (or their proxies, r² ≥ 0.8) in the whole
sample, in never-smokers, and in current smokers were cis-expression Quantitative Trait
Loci (cis-eQTLs) or methylation Quantitative Trait Loci (meQTLs). For eQTLs and meQTLs,
we used the browser Phenoscanner v2 (http://www.phenoscanner.medschl.cam.ac.uk/,
accessed on 18 October 2021), which combines several databases, e.g., the Genotype-Tissue
Expression project (GTEx, https://www.gtexportal.org/home/, accessed on 18 October
2021) for eQTLs, and BioSQLT and Gaunt's databases for meQTLs) and that includes e-QTL
data from many tissues [29–32]. Furthermore, functional annotations of these SNPs (or prox-
ies) were done using the HaploReg v4.1 tool (https://pubs.broadinstitute.org/mammals/
haploreg/haploreg.php, accessed on 18 October 2021). HaploReg annotates SNPs in terms
of colocalisation with regulatory elements, such as promoter and enhancer marks, DNase
I hypersensitivity sites, and transcription factor (TF) and protein-binding sites, based on
Roadmap Epigenomics data and Encyclopedia of DNA Elements data [33]. We checked the
potential deleteriousness of SNPs using the combined annotation-dependent depletion tool
(CADD v1.4, https://cadd.gs.washington.edu/, accessed on 1 April 2022). The CADD tool
scores the predicted deleteriousness of single nucleotide variants and insertion/deletions
variants in the human genome by integrating multiple annotations [34]. Note that a CADD score ≥ 15 indicates a deleterious effect of an SNP.

### 3. Results

The present analysis was carried out among adult participants (≥16 years old) at EGEA2 with available data on the FLOPs level, valid genotyped data, and asthma and tobacco smoking status. A total of 1216 adult participants were included in the analyses (see Figure S1 in Supplementary Materials).

Compared to the 355 adult participants not included in the analyses, the 1216 participants did not differ in terms of age, sex, and asthma status (data not shown).

#### 3.1. Characteristics of the Studied Population

The characteristics of the 1216 participants are presented in Table 1. The results are presented for the whole sample and according to asthma status (ever-asthmatics and never-asthmatics), to the current tobacco smoking status (never-smokers and current smokers), and to the study design (controls and cases/relatives). In the whole sample (mean age 43.3 years, 51% women), 44% had ever-asthma and 23% were current smokers. The geometric mean (GM) (Q1, Q3) of the FLOPs level was 92.3 (80, 105) RFU/mL.

Associations between the FLOPs level and the characteristics of the whole sample are presented in Table S1 (see Supplementary Materials). The FLOPs level was independently associated with age, sex, and smoking (all *p*-values < 5.0 × 10⁻³): it increased significantly with age, was significantly higher in women than in men, in ex-/current smokers (GM = 97.2 and 93.8 RFU/mL, respectively) as compared to never-smokers (GM = 89.1 RFU/mL), and increased significantly with lifelong cumulated quantity of cigarettes smoked. The geometric mean (GM) (Q1, Q3) of the FLOPs level was 93.4 (81, 107), 98.7 (88, 108), and 100.3 (88, 114) RFU/mL in participants with lifelong quantity of cigarettes smoked of <10, 10–20, and >20 pack-year, respectively (*p* < 1.0 × 10⁻⁴).

<table><caption>**Table 1.** The characteristics of the studied population.</caption><thead><tr><th></th><th>Whole Sample<br>(n = 1216)</th><th>Never-<br>Asthmatics<br>(n = 684)</th><th>Ever-<br>Asthmatics<br>(n = 532)</th><th>Never-<br>Smokers<br>(n = 604)</th><th>Current<br>Smokers<br>(n = 275)</th><th>Controls<br>(n = 243)</th><th>Cases/Relatives<br>(n = 973)</th></tr></thead><tbody><tr><td>Age, m ± sd</td><td>43.3 ± 16.4</td><td>46.3 ± 15.9</td><td>39.4 ± 16.4</td><td>42.7 ± 16.9</td><td>35.5 ± 14.1</td><td>47.2 ± 17.2</td><td>42.3 ± 16.1</td></tr><tr><td>Sex, women, n (%)</td><td>621 (51.1)</td><td>374 (54.7)</td><td>247 (46.4)</td><td>338 (56.0)</td><td>131 (47.6)</td><td>125 (51.4)</td><td>496 (51.0)</td></tr><tr><td>Current smoking status,<br>n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Never-smokers</td><td>604 (49.7)</td><td>342 (50.0)</td><td>262 (49.2)</td><td>604 (100)</td><td>-</td><td>114 (46.9)</td><td>490 (50.4)</td></tr><tr><td>Ex-smokers</td><td>337 (27.7)</td><td>202 (29.5)</td><td>135 (25.4)</td><td>-</td><td>-</td><td>70 (28.8)</td><td>267 (27.4)</td></tr><tr><td>Current smokers</td><td>275 (22.6)</td><td>140 (20.5)</td><td>135 (25.4)</td><td>-</td><td>275 (100)</td><td>59 (24.3)</td><td>216 (22.2)</td></tr><tr><td>Smoking, pack-year, n (%)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Never-smokers</td><td>604 (49.7)</td><td>342 (50.0)</td><td>262 (49.3)</td><td>604 (100)</td><td>-</td><td>114 (46.9)</td><td>490 (50.4)</td></tr><tr><td>&lt;10</td><td>384 (31.6)</td><td>197 (28.8)</td><td>187 (35.1)</td><td>-</td><td>186 (67.6)</td><td>75 (30.9)</td><td>309 (31.8)</td></tr><tr><td>10–20</td><td>111 (9.1)</td><td>68 (9.9)</td><td>43 (8.1)</td><td>-</td><td>44 (16.0)</td><td>23 (9.5)</td><td>88 (9.0)</td></tr><tr><td>&gt;20</td><td>117 (9.6)</td><td>77 (11.3)</td><td>40 (7.5)</td><td>-</td><td>45 (16.4)</td><td>31 (12.7)</td><td>86 (8.8)</td></tr><tr><td>BMI, kg·m<sup>-2</sup>, n (%)</td><td>n = 1201</td><td>n = 676</td><td>n = 525</td><td>n = 597</td><td>n = 272</td><td>n = 240</td><td>n = 961</td></tr><tr><td>≥30</td><td>118 (9.8)</td><td>66 (9.8)</td><td>52 (9.9)</td><td>56 (9.4)</td><td>16 (5.9)</td><td>20 (8.3)</td><td>98 (10.2)</td></tr><tr><td>IgE, IU/mL, m ± sd</td><td>n = 1214</td><td>n = 682</td><td>n = 532</td><td>n = 603</td><td>n = 274</td><td>n = 242</td><td>n = 972</td></tr><tr><td>White blood count</td><td>222 ± 453</td><td>134 ± 352</td><td>333 ± 537</td><td>198 ± 431</td><td>304 ± 528</td><td>116 ± 258</td><td>248 ± 487</td></tr><tr><td>Neutrophils, cells/mm<sup>3</sup>,<br>m ± sd</td><td>3986 ± 1391</td><td>3959 ± 1317</td><td>4020 ± 1481</td><td>3879 ± 1278</td><td>4314 ± 1535</td><td>3985 ± 1317</td><td>3986 ± 1410</td></tr><tr><td>Eosinophils, cells/mm<sup>3</sup>,<br>m ± sd</td><td>202 ± 155</td><td>168 ± 125</td><td>244 ± 179</td><td>203 ± 166</td><td>220 ± 162</td><td>160 ± 116</td><td>212 ± 162</td></tr><tr><td>Lung Function</td><td>n = 1197</td><td>n = 673</td><td>n = 524</td><td>n = 594</td><td>n = 272</td><td>n = 238</td><td>n = 959</td></tr><tr><td>FEV<sub>1</sub>, % predicted, m ± sd</td><td>103 ± 17.8</td><td>107 ± 16.3</td><td>97 ± 18.2</td><td>104 ± 17.5</td><td>100 ± 15.6</td><td>105 ± 15.4</td><td>102 ± 18.3</td></tr><tr><td>FVC, % predicted, m ± sd</td><td>110 ± 17.1</td><td>112 ± 17.5</td><td>108 ± 16.3</td><td>112 ± 17.1</td><td>107 ± 15.0</td><td>109 ± 15.3</td><td>111 ± 17.5</td></tr></tbody></table>
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th></th><th>Whole Sample<br>(n = 1216)</th><th>Never-Asthmatics<br>(n = 684)</th><th>Ever-Asthmatics<br>(n = 532)</th><th>Never-Smokers<br>(n = 604)</th><th>Current Smokers<br>(n = 275)</th><th>Controls<br>(n = 243)</th><th>Cases/Relatives<br>(n = 973)</th></tr></thead><tbody><tr><td>FEV<sub>1</sub> ≥ 80%, n (%)</td><td>1097 (91.7)</td><td>644 (95.7)</td><td>453 (86.5)</td><td>556 (93.6)</td><td>253 (93.0)</td><td>228 (95.8)</td><td>869 (90.6)</td></tr><tr><td>Chronic bronchitis, n (%)</td><td>n = 1205</td><td>n = 679</td><td>n = 526</td><td>n = 597</td><td>n = 274</td><td>n = 241</td><td>n = 964</td></tr><tr><td>Yes</td><td>114 (9.5)</td><td>39 (5.7)</td><td>75 (14.3)</td><td>47 (7.9)</td><td>40 (14.6)</td><td>19 (7.9)</td><td>95 (9.9)</td></tr><tr><td>FLOPs, RFU/mL, GM<br>(Q1, Q3)</td><td>92.3 (80, 105)</td><td>93.9 (82, 108)</td><td>90.4 (78, 102)</td><td>89.1 (77, 101)</td><td>93.8 (82, 107)</td><td>92.6 (81, 107)</td><td>92.3 (79, 105)</td></tr></tbody></table>

m, mean; sd, standard deviation; FEV₁, forced expiratory volume in one second; FVC, force vital capacity; GM, geometric mean; Q1, first quartile; and Q3, third quartile.

## 3.2. GWAS of the FLOPs Level

### 3.2.1. Whole Sample

Table 2 reports the results of the associations in the whole sample for the 10 SNPs showing the strongest signals. The Manhattan plot is available in Supplementary Materials, Figure S2A, and the Q-Q plot in Figure S3A shows that there was no inflation in the statistical test, with the genomic inflation factor estimated to 1.002. The top three SNPs were rs270404, located in the *BMP6* gene on chromosome 6p24.3 (*p* = 3.0 × 10⁻⁶); rs13223298, located upstream (from 2 kb apart) of the *BMPER* gene on chromosome 7p14.3 (*p*-value = 8.7 × 10⁻⁶); and rs491274, located in the intergenic region nearest *SEMA6D* (from 607 kb apart) genes on chromosome 15q21.1 (*p* = 8.9 × 10⁻⁶). The association analysis for these top three SNPs in the two independent sub-samples (controls vs. cases/relatives) showed no indication of heterogeneity (all *p*-values > 0.6, see Table S2 in Supplementary Materials).

The associations stratified by asthma status (never-asthmatics, ever-asthmatics) for the top three SNPs associated with the FLOPs level in the whole sample are reported in Table 3. No indication of heterogeneity was observed (all *p*-values ≥ 0.25).

An analysis using imputed SNPs spanning 500 kb on each side of each top SNP in the regions of the top three SNPs located in *BMP6*, near *BMPER* and near *SEMA6D*, confirmed the initial results (see Supplementary Materials: Figure S4A-C). Analyses of imputed SNPs in these regions found signals with similar or slightly improved significance levels at genotyped SNPs, and for at least two other imputed SNPs, which were close to and in strong linkage disequilibrium (LD, r² > 0.8) with the genotyped top SNP (Figure S4A-C).

<table><caption>**Table 2.** The top 10 SNPs associated with the FLOPs level in 1216 adults from the EGEA study.</caption><thead><tr><th>Chr</th><th>Gene</th><th>Nearest Gene</th><th>Genomic Location</th><th>Marker</th><th>Position bp (hg38)</th><th>Band</th><th>A1/A2</th><th>EAF</th><th>Beta ± se</th><th>p</th></tr></thead><tbody><tr><td>6</td><td>BMP6</td><td>TXNDC5</td><td>Intronic</td><td>rs270404</td><td>7,757,141</td><td>p24.3</td><td>A/G</td><td>0.41</td><td>-0.20 ± 0.04</td><td>3.0 × 10<sup>-6</sup></td></tr><tr><td>7</td><td></td><td>BMPER</td><td>3'-UTR</td><td>rs13223298</td><td>34,158,658</td><td>p14.3</td><td>G/T</td><td>0.08</td><td>-0.34 ± 0.08</td><td>8.7 × 10<sup>-6</sup></td></tr><tr><td>15</td><td></td><td>SEMA6D</td><td>Intergenic</td><td>rs491274</td><td>46,577,328</td><td>q21.1</td><td>A/G</td><td>0.09</td><td>-0.31 ± 0.07</td><td>8.9 × 10<sup>-6</sup></td></tr><tr><td>1</td><td>RGL1</td><td>APOBEC4</td><td>Intronic</td><td>rs6664058</td><td>183,687,143</td><td>q25.3</td><td>C/T</td><td>0.36</td><td>0.21 ± 0.05</td><td>1.2 × 10<sup>-5</sup></td></tr><tr><td>7</td><td>PKD1L1</td><td>TNS3</td><td>Intronic</td><td>rs10276437</td><td>47,766,479</td><td>p12.3</td><td>C/T</td><td>0.78</td><td>0.21 ± 0.05</td><td>1.3 × 10<sup>-5</sup></td></tr><tr><td>14</td><td></td><td>VRK1</td><td>Intergenic</td><td>rs4905587</td><td>97,213,901</td><td>q32.2</td><td>G/T</td><td>0.12</td><td>0.31 ± 0.07</td><td>1.4 × 10<sup>-5</sup></td></tr><tr><td>1</td><td>RGL1</td><td>APOBEC4</td><td>Intronic</td><td>rs6424909</td><td>183,727,521</td><td>q25.3</td><td>A/G</td><td>0.36</td><td>0.21 ± 0.05</td><td>1.5 × 10<sup>-5</sup></td></tr><tr><td>16</td><td></td><td>HS3ST6</td><td>5'-UTR</td><td>rs344363</td><td>1,922,547</td><td>p13.3</td><td>C/T</td><td>0.17</td><td>-0.26 ± 0.06</td><td>1.7 × 10<sup>-5</sup></td></tr><tr><td>20</td><td>PMEPA1</td><td>ZBP1</td><td>Intronic</td><td>rs6025728</td><td>57,697,562</td><td>q13.31</td><td>C/T</td><td>0.63</td><td>0.20 ± 0.05</td><td>2.0 × 10<sup>-5</sup></td></tr><tr><td>12</td><td>CACNA1C</td><td>FKBP4</td><td>Intronic</td><td>rs4765961</td><td>2,559,306</td><td>p13.33</td><td>C/T</td><td>0.82</td><td>0.23 ± 0.05</td><td>2.2 × 10<sup>-5</sup></td></tr></tbody></table>

Chr, chromosome; A1/A2, baseline/effect allele; and EAF, effect allele frequency estimated from the reference panel 1000 G (European population). Beta and standard error (se) were estimated using the Gaussian linear model, taking into account the EGEA family structure and adjusted for principal components.
<table><caption>**Table 3.** A stratified analysis according to asthma status for the top three SNPs in the whole sample.</caption><thead><tr><th rowspan="2">Chr</th><th rowspan="2">Gene</th><th rowspan="2">Nearest Gene</th><th rowspan="2">Genomic Location</th><th rowspan="2">Marker</th><th rowspan="2">A1/A2</th><th rowspan="2">EAF</th><th colspan="2">Never-Asthmatics (n = 684)</th><th colspan="2">Ever-Asthmatics (n = 532)</th><th colspan="2">Homogeneity Test</th></tr><tr><th>Beta ± se</th><th>p</th><th>Beta ± se</th><th>p</th><th>Khi<sup>2</sup></th><th>p</th></tr></thead><tbody><tr><td>6</td><td>BMP6</td><td>TXNDC5</td><td>Intronic</td><td>rs270404</td><td>A/G</td><td>0.41</td><td>-0.20 ± 0.06</td><td>5.1 × 10<sup>-4</sup></td><td>-0.20 ± 0.06</td><td>5.4 × 10<sup>-4</sup></td><td>0.01</td><td>0.91</td></tr><tr><td>7</td><td>BMPER</td><td>3'-UTR</td><td></td><td>rs13223298</td><td>G/T</td><td>0.08</td><td>-0.42 ± 0.10</td><td>4.5 × 10<sup>-5</sup></td><td>-0.23 ± 0.12</td><td>5.5 × 10<sup>-2</sup></td><td>1.32</td><td>0.25</td></tr><tr><td>15</td><td>SEMA6D</td><td>Intergenic</td><td></td><td>rs491274</td><td>A/G</td><td>0.09</td><td>-0.36 ± 0.08</td><td>2.7 × 10<sup>-5</sup></td><td>-0.23 ± 0.11</td><td>4.1 × 10<sup>-2</sup></td><td>0.75</td><td>0.39</td></tr></tbody></table>

Chr, chromosome; A1/A2, baseline/effect allele; and EAF, effect allele frequency estimated from the reference panel 1000 G (European population). Beta and standard error (se) were estimated using Gaussian linear model, taking into account EGEA family structure and adjusted for principal components.

### 3.2.2. In Never-Smokers and in Current Smokers

Table 4 presents the associations with FLOPs for the top 10 SNPs in never-smokers and in current smokers. Manhattan plots are available for the two GWAS in Supplementary Materials; Figure S2B,C; and the Q-Q plots in Figure S3B,C and show that there was no inflation in the statistical test for the two GWAS, with genomic inflation factors estimated to 1.006 and 1.03, respectively.

<table><caption>**Table 4.** The GWAS of FLOPs: the top 10 SNPs in never-smokers and in current smokers.</caption><thead><tr><th>Chr</th><th>Gene</th><th>Nearest Gene</th><th>Genomic Location</th><th>Marker</th><th>Position bp (hg38)</th><th>Band</th><th>A1/A2</th><th>EAF</th><th>Beta ± se</th><th>p</th></tr></thead><tbody><tr><td colspan="11" style="text-align: left; font-weight: bold;">Never-smokers (n = 604)</td></tr><tr><td>6</td><td>COL21A1</td><td>DST</td><td>Intronic</td><td>rs17823624</td><td>56,348,021</td><td>p12.1</td><td>A/G</td><td>0.92</td><td>0.44 ± 0.08</td><td>2.3 × 10<sup>-7</sup></td></tr><tr><td>15</td><td>GABRG3</td><td>GABRA5</td><td>Intronic</td><td>rs6606856</td><td>27,022,887</td><td>q12</td><td>C/T</td><td>0.70</td><td>-0.28 ± 0.05</td><td>4.1 × 10<sup>-7</sup></td></tr><tr><td>5</td><td></td><td>NUDT12</td><td>Intergenic</td><td>rs2962642</td><td>104,131,010</td><td>q21.2</td><td>A/G</td><td>0.24</td><td>0.30 ± 0.06</td><td>4.4 × 10<sup>-7</sup></td></tr><tr><td>10</td><td>PAOX</td><td>MTG1</td><td>Intronic</td><td>rs6537600</td><td>133,391,295</td><td>q26.3</td><td>C/T</td><td>0.88</td><td>0.40 ± 0.08</td><td>6.5 × 10<sup>-7</sup></td></tr><tr><td>5</td><td></td><td>NUDT12</td><td>Intergenic</td><td>rs7725285</td><td>104,111,482</td><td>q21.2</td><td>G/T</td><td>0.75</td><td>-0.29 ± 0.06</td><td>1.5 × 10<sup>-6</sup></td></tr><tr><td>10</td><td>PAOX</td><td>MTG1</td><td>Intronic</td><td>rs10776679</td><td>133,389,090</td><td>q26.3</td><td>C/T</td><td>0.05</td><td>-0.49 ± 0.10</td><td>1.6 × 10<sup>-6</sup></td></tr><tr><td>8</td><td>DEFB135</td><td>DEFB136</td><td>Intronic</td><td>rs6985349</td><td>11,982,562</td><td>p23.1</td><td>C/T</td><td>0.06</td><td>-0.43 ± 0.09</td><td>2.0 × 10<sup>-6</sup></td></tr><tr><td>8</td><td>DEFB135</td><td>DEFB136</td><td>Intronic</td><td>rs7004833</td><td>11,982,502</td><td>p23.1</td><td>A/G</td><td>0.06</td><td>-0.43 ± 0.09</td><td>2.0 × 10<sup>-6</sup></td></tr><tr><td>3</td><td></td><td>ZNF385D</td><td>Intronic</td><td>rs1391857</td><td>20,857,452</td><td>p24.3</td><td>A/G</td><td>0.61</td><td>-0.27 ± 0.06</td><td>2.5 × 10<sup>-6</sup></td></tr><tr><td>10</td><td>ECHS1</td><td>PAOX</td><td>Missense</td><td>rs1049951</td><td>133,370,622</td><td>q26.3</td><td>A/G</td><td>0.06</td><td>-0.41 ± 0.09</td><td>3.6 × 10<sup>-6</sup></td></tr><tr><td colspan="11" style="text-align: left; font-weight: bold;">Current smokers (n = 275)</td></tr><tr><td>6</td><td>CRYBG1</td><td>ATG5</td><td>Intronic</td><td>rs3851212</td><td>106,375,664</td><td>q21</td><td>A/G</td><td>0.94</td><td>-0.81 ± 0.13</td><td>2.4 × 10<sup>-9</sup></td></tr><tr><td>12</td><td>COL2A1</td><td>TMEM106C</td><td>Intronic</td><td>rs1793958</td><td>47,998,650</td><td>q13.11</td><td>A/G</td><td>0.61</td><td>-0.40 ± 0.08</td><td>4.7 × 10<sup>-7</sup></td></tr><tr><td>12</td><td></td><td>PTPRO</td><td>Intergenic</td><td>rs17174795</td><td>15,603,555</td><td>p12.3</td><td>G/T</td><td>0.87</td><td>-0.62 ± 0.12</td><td>9.2 × 10<sup>-7</sup></td></tr><tr><td>15</td><td>ISG20</td><td>AEN</td><td>Intronic</td><td>rs8041687</td><td>88,655,329</td><td>q26.1</td><td>A/G</td><td>0.07</td><td>0.56 ± 0.11</td><td>1.1 × 10<sup>-6</sup></td></tr><tr><td>3</td><td>CMC1</td><td>AZI2</td><td>Intronic</td><td>rs7641491</td><td>28,301,843</td><td>p24.1</td><td>A/G</td><td>0.53</td><td>0.39 ± 0.08</td><td>2.8 × 10<sup>-6</sup></td></tr><tr><td>3</td><td>CMC1</td><td>AZI2</td><td>Intronic</td><td>rs13085075</td><td>28,309,712</td><td>p24.1</td><td>C/T</td><td>0.48</td><td>-0.39 ± 0.08</td><td>3.1 × 10<sup>-6</sup></td></tr><tr><td>7</td><td></td><td>CREB5</td><td>Intergenic</td><td>rs10228137</td><td>28,848,469</td><td>p14.3</td><td>A/C</td><td>0.81</td><td>-0.43 ± 0.09</td><td>4.3 × 10<sup>-6</sup></td></tr><tr><td>2</td><td></td><td>LRP1B</td><td>Intergenic</td><td>rs961109</td><td>142,396,452</td><td>q22.2</td><td>C/T</td><td>0.91</td><td>0.66 ± 0.14</td><td>4.6 × 10<sup>-6</sup></td></tr><tr><td>4</td><td>TENM3</td><td>DCTD</td><td>Intronic</td><td>rs4557308</td><td>182,734,597</td><td>q35.1</td><td>A/G</td><td>0.26</td><td>0.41 ± 0.09</td><td>5.0 × 10<sup>-6</sup></td></tr><tr><td>13</td><td></td><td>ATP7B</td><td>5'-UTR</td><td>rs4943040</td><td>51,919,816</td><td>q14.3</td><td>C/T</td><td>0.55</td><td>0.38 ± 0.08</td><td>5.2 × 10<sup>-6</sup></td></tr></tbody></table>

Chr, chromosome; A1/A2, baseline/effect allele; and EAF, effect allele frequency estimated from the reference panel 1000 G (European population). Beta and standard error (se) were estimated using Gaussian linear model, taking into account EGEA family structure and adjusted for principal components.

In never-smokers, the top three SNPs (all *p*-values < 5.0 × 10⁻⁷) were rs17823624 located in the COL21A1 gene on the chromosome 6p12.1 (*p* = 2.3 × 10⁻⁷), rs6606856 located in GABRG3 gene on chromosome 15q12 (*p* = 4.1 × 10⁻⁷), and rs2962642 located in an intergenic region near NUDT12 (from 568 kb apart) on chromosome 5q21.2 (*p* = 4.4 × 10⁻⁷). For these top three SNPs, association analysis in the two independent sub-samples (controls vs. cases/relatives) showed no indication of heterogeneity (all *p*-values > 0.6, see Table S3 in Supplementary Materials). Besides that, association analysis for these top SNPs yielded
similar results in never-asthmatics and in ever-asthmatics, with no indication of heterogeneity ($p \ge 0.10$, Table 5). Note that none of the top three SNPs found in never-smokers showed indication of association in current smokers (all $p$-values > 0.10).

In current smokers, the top three SNPs were rs3851212 located in *CRYBG1* on chromosome 6q21 ($p = 2.4 \times 10^{-9}$, exceeding the significance level of $1.3 \times 10^{-7}$), rs1793958 located in *COL2A1* on chromosome 12q13.11 ($p = 4.7 \times 10^{-7}$), and rs17174795 located upstream *PTPRO* (from 1.4 kb apart) on chromosome 12 p12.3 ($p = 9.2 \times 10^{-7}$). For these top three SNPs, association analysis in the two independent sub-samples (controls vs. cases/relatives) showed no indication of heterogeneity (all $p$-values ≥ 0.4, see Table S3 in Supplementary Materials). No heterogeneity of association signals was detected according to asthma status (all $p$-values ≥ 0.7, except for rs17174795 with $p$-value = 0.05, but not significant after correction for multiple testing; see Table 5). The top three SNPs found in current smokers showed no indication of association in never-smokers ($p > 0.20$) or only a weak signal ($p = 0.05$).

<table><caption>**Table 5.** The stratified analysis according to asthma status for the top three SNPs, in never-smokers and in current smokers.</caption><thead><tr><th rowspan="2">Chr</th><th rowspan="2">Gene</th><th rowspan="2">Nearest Gene</th><th rowspan="2">Genomic Location</th><th rowspan="2">Marker</th><th rowspan="2">Position bp (hg38)</th><th rowspan="2">A1/A2</th><th rowspan="2">EAF</th><th colspan="2">Never-Asthmatics</th><th colspan="2">Ever-Asthmatics</th><th colspan="2">Homogeneity Test</th></tr><tr><th>Beta ± se</th><th>p</th><th>Beta ± se</th><th>p</th><th>khi<sup>2</sup></th><th>p</th></tr></thead><tbody><tr><td></td><td>Never-smokers (n = 604)</td><td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2">n = 342</td><td colspan="2">n = 262</td><td></td><td></td></tr><tr><td>6</td><td>COL21A1</td><td>DST</td><td>Intronic</td><td>rs17823624</td><td>56 348 021</td><td>A/G</td><td>0.92</td><td>0.46 ± 0.11</td><td>2.9 × 10<sup>-5</sup></td><td>0.43 ± 0.13</td><td>1.1 × 10<sup>-3</sup></td><td>0.07</td><td>0.79</td></tr><tr><td>15</td><td>GABRG3</td><td>GABRA5</td><td>Intronic</td><td>rs6606856</td><td>27 022 887</td><td>C/T</td><td>0.70</td><td>-0.29 ± 0.08</td><td>2.4 × 10<sup>-4</sup></td><td>-0.30 ± 0.08</td><td>1.7 × 10<sup>-4</sup></td><td>0.00</td><td>0.97</td></tr><tr><td>5</td><td></td><td>NUDT12</td><td>Intergenic</td><td>rs2962642</td><td>104 131 010</td><td>A/G</td><td>0.24</td><td>0.36 ± 0.07</td><td>2.0 × 10<sup>-6</sup></td><td>0.22 ± 0.09</td><td>1.5 × 10<sup>-2</sup></td><td>1.89</td><td>0.17</td></tr><tr><td></td><td>Current smokers (n = 275)</td><td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2">n = 140</td><td colspan="2">n = 135</td><td></td><td></td></tr><tr><td>6</td><td>CRYBG1</td><td>ATG5</td><td>Intronic</td><td>rs3851212</td><td>106 375 664</td><td>A/G</td><td>0.94</td><td>-0.79 ± 0.22</td><td>5.5 × 10<sup>-4</sup></td><td>-0.85 ± 0.16</td><td>3.2 × 10<sup>-7</sup></td><td>0.08</td><td>0.78</td></tr><tr><td>12</td><td>COL2A1</td><td>TMEM106C</td><td>Intronic</td><td>rs1793958</td><td>47 998 650</td><td>A/G</td><td>0.61</td><td>-0.38 ± 0.12</td><td>1.7 × 10<sup>-3</sup></td><td>-0.42 ± 0.10</td><td>4.9 × 10<sup>-5</sup></td><td>0.09</td><td>0.77</td></tr><tr><td>12</td><td></td><td>PTPRO</td><td>Intergenic</td><td>rs17174795</td><td>15 603 555</td><td>G/T</td><td>0.87</td><td>-0.85 ± 0.18</td><td>5.1 × 10<sup>-6</sup></td><td>-0.36 ± 0.16</td><td>2.7 × 10<sup>-2</sup></td><td>3.86</td><td>0.05</td></tr></tbody></table>

Chr, chromosome; A1/A2, baseline/effect allele; and EAF, effect allele frequency estimated from the reference panel 1000 G (European population). Beta and standard error (se) were estimated using Gaussian linear model, taking into account EGEA family structure and adjusted for principal components.

Analysis using imputed SNPs spanning 500 kb on each side of each top SNP in the regions of the top three SNPs in both never-smokers and current smokers confirmed the initial results, with a similar significance level as those observed with genotyped SNPs (Figures S5A–C and S6A–C in Supplementary Materials). Analyses of imputed data in the region around two of the top six SNPs found additional signals at imputed SNPs close to and in strong linkage disequilibrium (LD, $r^2 > 0.8$), with the genotyped top SNPs, 12 for rs2962642 located near *NUDT12* with similar significance level (Figure S5C) and two for rs17174795 located near *PTPRO* with improved significance level (Figure S6C). These results also supported the initial findings for these top two SNPs.

### 3.3. eQTLs, meQTLs, and Functional Annotations

Using the browser Phenoscanner v2 (http://www.phenoscanner.medschl.cam.ac.uk/, accessed on 18 October 2021), we found that top two SNPs, one in never-smokers (rs17823624 in *COL21A1*) and one in current smokers (rs1793958 in *COL2A1*), were associated with gene expression in a whole blood sample from subject of European ancestry (Table S4 in Supplementary Materials). The SNP rs17823624 was associated with gene expression of *DST* ($p$-value = $2.0 \times 10^{-15}$), while the SNP rs1793958 was associated with the expression of five genes belonging to the 12q13.11 region: *OR10AD1*, *PFKM*, *SENP1*, *TMEM106C*, and *VDR* (all $p$-values < $2.5 \times 10^{-8}$). No eQTL was found for the other top SNPs. Furthermore, we found from 1 to 26 CpG sites considering all top three SNPs of GWAS in the whole sample, in never-smokers and in current smokers (See Table S5 in Supplementary Materials). Most CpG sites were located near or in the same gene as the associated SNP. The SNP rs13223298 in *BMPER* was found to be more associated with the
methylation level of one CpG site located on another chromosome than *BMPER*, in the *PTPN22* gene on chromosome 1p13.2 (*p*-value = 9.3 × 10<sup>-8</sup>, not significant after correction for multiple testing).

Note that a proxy of rs3851212 (the top SNP in current smokers located in *CRYBG1*) rs79231630 had a CADD score equal to 14.5. Detailed results for CADD scores are presented in Table S6.

Using the functional annotation tool HaploReg-v4.1, we found that all top three SNPs (or their proxies) evidenced respectively in the whole sample, in never-smokers and in current smokers, mapped to marks of active regulatory elements, including cells from heart, lung, kidney, breast, and brain. Detailed results for functional annotations are presented in Table S6 (see Supplementary Materials).

**4. Discussion**

This first genetic study on the FLOPs level identified several variants, among which those located in *BMP6*, near *BMPER* and between *SQOR* and *SEMA6D* were the most strongly associated with this biomarker in the whole sample. Stratified analyses on tobacco smoking status identified other genetic variants: among them, the top three SNPS in never-smokers located in *COL21A1*, in *GABRG3*, and near *NUDT12*, and the top three SNPS in current smokers located in *CRYBG1*, in *COL2A1*, and near *PTPRO*.

Our study is based on the hypothesis that GWAS of FLOPs may provide new insights into the pathophysiology of chronic diseases related to the OS pathway. The GWAS analyses we performed were based on the EGEA study, whose participants had extensive clinical, genetic, biological, and environmental characterisation. To our knowledge, there was no other epidemiological study with such data for a replication sample. Interestingly, all our association results were supported by consistent results observed in controls and cases/relative sub-samples. Furthermore, analyses of imputed data in the region around each top SNP confirmed our initial association results obtained with genotyped data. However, all our findings should be validated/replicated in other independent cohorts.

Due to the ascertainment mode of families in the EGEA study, i.e., through asthmatic participants, and the involvement of the OS pathway in the pathophysiology of asthma, we repeated our analyses in ever- and never-asthmatics in order to evaluate the associations independently of the disease. The results were consistent between never- and ever-asthmatics, which showed the independence of our results from the disease. Furthermore, we verified that any of our top SNPs were associated with lung function or adult-onset asthma in EGEA sample [19]. All these results suggest that our main results are not driven by asthma.

In the whole population, the strongest association signals were observed for rs270404 located in *BMP6* and rs13223298 located near *BMPER* (i.e., 2.2 kb from that gene). *BMP6* and *BMPER* were reported to interact physically in a functional study [35]. In line with this result, we tested the effect of the statistical interaction between these SNPs on FLOPs and found a borderline significant interaction (*p*-value = 0.07). *BMP6* (Bone Morphogenetic Protein 6) encodes a secreted ligand of the transforming growth factor-beta (TGF-beta) superfamily of proteins, and *BMPER* (BMP Binding Endothelial Regulator) encodes a secreted protein that interacts with and inhibits the bone morphogenetic protein (BMP) function. It is noteworthy that these two genes belong to the biological process "regulation of pathway restricted SMAD protein phosphorylation" pathway (GO:006093) that is involved in the TGF-beta receptor signalling pathways [36]. The role of TGF-beta has been discussed in chronic asthma, as a potent fibrogenic growth factor overexpressed in the asthmatic lung [37]. Moreover, *BMPER* belongs to the biological process "immune response" pathway (GO:0006954) [38]. From the GWAS Catalog [39], we found that *BMP6* was associated with FVC [40–42]; FEV₁ [42]; and, to a lesser extent, chronic obstructive pulmonary disease [43] and small cell lung carcinoma [44]. In previous GWAS, associations were reported between *BMPER* and FVC [40], FEV₁ [45], and other chronic diseases such as Alzheimer's disease [46] and metastatic colorectal cancer [47]. Moreover, the top two SNPs in *BMP6* and near the *BMPER* map to marks of active regulatory elements in heart, lung, brain, breast,
and kidney tissues and the top SNP near *BMPER* was associated with the methylation level of one CpG site located in *PTPN22* (Protein Tyrosine Phosphatase Non-Receptor Type 22), a gene involved in “NF-Kappa B signalling” and “immune response” pathways.

The third strongest association signal was observed for rs491274 located nearest *SEMA6D* (i.e., 607 kb apart). *SEMA6D* (Semaphorin 6D) was found to be associated with parental longevity [48,49] and lung carcinoma [44] in previous GWAS.

As cigarette smoke is the most important exogenous source of ROS, we performed GWAS separately in two contrasted sub-groups according to smoking status and identified specific association signals in each sub-group. In never-smokers, the top SNP was rs1782324 located in *COL21A1* (Collagen Type XXI Alpha 1 Chain). In previous GWAS, *COL21A1* showed associations with lung function [40] and to a lesser extend with allergic sensitisation [50] and small cell lung carcinoma [44]. We also found that rs17823624 was eQTL of *DST* (Dystonine), a gene close to *COL21A1*, for which variants were found to be associated with lung function [40,42] and Alzheimer’s disease [51]. The next top SNP rs6606856 was located in *GABRG3* (Gamma-Aminobutyric Acid Type A Receptor Subunit Gamma3), a gene involved in the “response to drug” biological pathway (GO:0042493). This gene was shown to be associated with gene methylation in the lung tissue of smokers, as reported in a previous GWAS [52]. An association between *GABRG3* and several chronic diseases, including Alzheimer’s disease [53], ovarian carcinoma [54,55], and non-melanoma carcinoma [56], was also reported in other previous GWAS. Finally, the third highest SNP rs2962642 was located near (i.e., 568 kbp apart) to *NUDT12* (Nudix Hydrolase 12). This gene was involved in the “NADH metabolic process” biological pathway (GO:0006734), and was shown to be associated with longevity [57] and smoking behaviour [58] in previous GWAS. Furthermore, proxies of rs2962642 map onto the regulatory motifs altered for histone deacetylase 2 (HDAC2), whose activity is regulated by oxidative stress.

In current smokers, the top SNP, rs3851212, was located in *CRYBG1*, which exceeded the genome-wide significance threshold level of 4.3 × 10⁻⁸ when accounting for the three GWAS. The role of *CRYBG1* (crystallin beta-gamma domain containing 1), also called *AIM1* (absent in melanoma 1 protein) in malignant melanoma, is well-known [59]. To note, rs3851212 is located 50 kb from *ATG5* (autophagy-related 5), which belongs to the biological pathway “immune system process” (GO:0002376) and is involved in mitochondrial quality control after oxidative damage, and in subsequent cellular longevity. In previous GWAS, *ATG5* was found to be associated with allergic diseases [60], including asthma [61–64], and with several other chronic diseases such as systemic lupus erythematous [65–68], rheumatoid arthritis [69,70], and multiple myeloma [71]. Furthermore, note that a proxy of rs3851212—rs79231630—has a CADD score close to 15, indicating deleterious effect of the SNP. The next top SNP is rs1793958, located in *COL2A1* (collagen type II alpha 1 chain), a gene involved in “regulation of immune response” biological process (GO:0050776). *COL2A1* was found to be associated with rheumatoid arthritis [72] and with prostate carcinoma [73–75] in previous GWAS. On the other hand, the third highest SNP rs17174795 is located 1.4 kb apart from *PTPRO* (Protein Tyrosine Phosphatase Receptor type O), which has been suggested as a candidate tumour suppressor via the NF-Kappa B signalling pathway [76,77], and the transcription factor NF-Kappa B plays a central role in inflammatory airway diseases such as asthma [78].

None of the top signals found in one sub-group of smoking status were found in the other sub-group, nor in the whole sample, showing that, as we hypothesised, accounting for smoking status may help one to identify loci not found in the whole sample. These results are likely explained by the existing interactions between the environment (here smoking) and genes that lead to “up” or “down” regulation of the pathways that influence the level of FLOPs.

None of the top signals found in the whole sample were found in the two contrasted sub-groups according to smoking status, which is an interesting result. Indeed, these analyses were carried out with the objective to identify genetic loci that could have been missed in the whole sample as smoking is the main environmental source of OS and is
associated with FLOPs. The two GWAS in groups contrasted by smoking revealed additional
genetic loci to those found in the whole sample.

Overall, many of the top SNPs identified by the three GWAS are located in regions
comprising promising candidate genes. Among them, *BMP6*, *BMPER*, *GABRG3*, and *ATG5*
are the most relevant because of both their link to biological pathways related to OS and
their association with several chronic diseases, for which the role of OS in their patho-
physiology has been pointed out. *BMP6* and *BMPER* are of particular interest due to their
involvement in the same biological pathways related to OS and their functional interaction.

**5. Conclusions**

In conclusion, the present study identified, for the first time, new and promising
candidate genes associated with the FLOPs level potentially involved in the pathophysiology
of chronic diseases through their link with the oxidative stress pathway. Although further
studies are needed to replicate these findings, this work highlights the interest in performing
genome-wide analyses of biomarkers to identify new genes and potential mechanisms
related to specific pathways common to chronic diseases.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox11050802/s1. References [15,16,79] are cited in the supplementary materials. The description of the EGEA study; Figure S1: a flow chart of the studied population; Figure S2: a Manhattan plot of the GWAS results of the FLOPs levels (A) in the whole sample (n = 1216), (B) in never-smokers (n = 604), and (C) in current smokers (n = 275); Figure S3: a quantile-quantile (QQ) plot of the GWAS results of the FLOPs levels (A) in the whole sample (n = 1216), (B) in never-smokers (n = 604), and (C) in current smokers (n = 275); Figure S4: a regional plot of the association results using imputed genetic data for the top three SNPs in the whole sample (n = 1216), the region around rs270404 (A), the region around rs13223298 (B), and the region around rs491274 (C); Figure S5: a regional plot of the association results using imputed genetic data for the top three SNPs in never-smokers (n = 604), the region around rs17823624 (A), the region around rs6606856 (B), and the region around rs2962642 (C); Figure S6: the regional plot of association results using imputed genetic data for the top three SNPs in current smokers (n = 275), the region around rs3851212 (A), the region around rs1793958 (B), and the region around rs17174795 (C); Table S1: the association between the FLOPs level and the characteristics of the studied population (n = 1216); Table S2: the top three SNPs in the whole sample: the consistency of the results in two independent sub-samples; Table S3: the top three SNPs in never-smokers and in current smokers: the consistency of the results in two independent sub-samples; Table S4: the results from the eQTL browser Phenoscanner v2; Table S5: the meQTLs for the top three-SNPs identified in GWAS in the whole sample, in never-smokers and in current smokers; Table S6: Regulatory elements for the top three SNPs (and proxies with r² > 0.80) identified in GWAS in the whole sample, in never-smokers and in current smokers.

**Author Contributions:** Conceptualisation, L.O., R.N. and M.-H.D.; data curation, L.O., P.M.-J. and M.-H.D.; formal analysis, L.O., P.M.-J. and M.A.; methodology, L.O., O.D., R.N. and M.-H.D.; project administration, R.N. and M.-H.D.; resources, L.O., P.M.-J., H.M., E.B., F.D., R.M., F.Z., R.N. and M.-H.D.; software, L.O., P.M.-J. and H.M.; supervision, R.N. and M.-H.D.; validation, L.O., R.N. and M.-H.D.; visualisation, L.O., R.N. and M.-H.D.; writing—original draft preparation, L.O., R.N. and M.-H.D.; and writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** The French National Research Program for Environmental and Occupational Health of Anses (EST/2017/1/158); the National Hospital program of clinical research (PHRC-National 2012, EvAdA); the National Research Agency—Health Environment, Health-Work Program (ANR-CES-2009, BIO2NEA); the Fonds AGIR pour les maladies chroniques; and the Region Hauts de France.
**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki. Approvals were obtained from the relevant Ethics Committees and the Institutional Review Board Committees: INSERM, RBM 'Recherche BioMédicale' (no RBM 91-005, 08/1991; and no RBM 01-11, 02/2002); CNIL 'Commission Nationale de l'Informatique et des Libertés' (no 109 427, 04/1990; and no 900 198, 10/2000); the Institutional Review Board Committees of Cochin Port-Royal Hospital and Necker-Enfants Malades Hospital, Paris, France (no 01-07-07, 09/2001; no 04-05-03, 11/2004; no 04-11-13, 11/2004; and no 04-11-18, 12/2004); and DGS 'Direction Générale de la Santé' (no DGS 910 048, 07/1991; and no DGS 2002/0106, 02/2002).

**Informed Consent Statement:** Written informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Due to third-party restrictions, EGEA data are not publicly available. Please see the following URL for more information: https://egeanet.vjf.inserm.fr/index.php/en/ contacts-en, accessed on 17 February 2022. Interested researchers should contact egea.cohorte@inserm.fr with further questions regarding data access.

**Acknowledgments:** The EGEA cooperative group. Coordination: V Siroux (epidemiology, PI since 2013); F Demenais (genetics); I Pin (clinical aspects); R Nadif (biology); and F Kauffmann (PI 1992-2012). Respiratory epidemiology: Inserm ex-U 700, Paris: M Korobaeff (Egea1) and F Neukirch (Egea1); Inserm ex-U 707, Paris: I Annesi-Maesano (Egea1-2); Inserm U 1018, Villejuif: O Dumas, F Kauffmann, N Le Moual, R Nadif, MP Oryszczyn (Egea1-2), and R Varraso; Inserm U 1209 Grenoble: J Lepeule, and V Siroux. Genetics: Inserm ex-U 393, Paris: J Feingold; Inserm UMR 1124, Paris: E Bouzigon, MH Dizier, and F Demenais; and CNG, Evry: I Gut (now CNAG, Barcelona, Spain), M Lathrop (now Univ McGill, Montreal, Canada). Clinical centres: Grenoble: I Pin and C Pison; Lyon: D Ecochard (Egea1), F Gormand, and Y Pacheco; Marseille: D Charpin (Egea1) and D Vervloet (Egea1-2); Montpellier: J Bousquet; Paris Cochin: A Lockhart (Egea1) and R Matran (now in Lille); Paris Necker: E Paty (Egea1-2) and P Scheinmann (Egea1-2); Paris-Trousseau: A Grimfeld (Egea1-2) and J Just. Data management and quality: Inserm ex-U155, Paris: J Hochez (Egea1); Inserm U 1018, Villejuif: N Le Moual and L Orsi; Inserm ex-U780, Villejuif: C Ravault (Egea1-2); Inserm ex-U794, Evry: N Chateigner (Egea1-2); Inserm UMR 1124, Paris: H Mohamdi; and Inserm U1209, Grenoble: A Boudier and J Quentin (Egea1-2). The authors thank all those who participated in the setting of the study and in the various aspects of the examinations involved: interviewers; technicians for lung function testing and skin prick tests, blood sampling, and IgE determinations; coders; those involved in quality control, data, and sample management; and all those who supervised the study in all centres. The authors are grateful to the three CIC-Inserm of Necker, Grenoble, and Marseille, who supported the study and in which participants were examined. They are also grateful to the biobanks in Lille (CIC Inserm), and at Annemasse (Etablissement Français du sang), where biological samples are stored. They are indebted to all the individuals who participated, without whom the study would not have been possible. We also thank P. Dessen for his helpful suggestions regarding genes bioinformatics applications.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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