Article published online: 2022-03-10 THIEME Stroke, Systemic or Venous Thromboembolism 1027 Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk Sonia Lopez1 Angel Martinez-Perez1 Alba Rodriguez-Rius1 Ana Viñuela2 Andrew A. Brown3 Laura Martin-Fernandez 1,4,5,6 Noelia Vilalta7 Marc Arús7 Nikolaos I. Panousis8,9 Alfonso Buil10 Maria Sabater-Lleal1,11 Juan Carlos Souto7 Emmanouil T. Dermitzakis9 Jose Manuel Soria1 1 Genomics of Complex Diseases Unit, Research Institute Hospital de Address for correspondence Sonia Lopez, PhD, Genomics of Complex la Santa Creu i Sant Pau, IIB Sant Pau, Barcelona, Spain Diseases Unit, Research Institute Hospital de la Santa Creu i Sant Pau, 2 Biosciences Institute, Faculty of Medicine, Newcastle University, IIB Sant Pau, Sant Quintí 77-79, 3rd Floor, 08041 Barcelona, Spain Newcastle Upon Tyne, United Kingdom (e-mail: slopezm@santpau.cat). 3 Population Health and Genomics, University of Dundee, Dundee, Scotland, United Kingdom 8 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, 4 Fundación Española de Trombosis y Hemostasia (FETH), Madrid, Spain South Cambridgeshire, United Kingdom 5 Congenital Coagulopathies Laboratory, Banc de Sang i Teixits, 9 Department of Genetic Medicine and Development, University of Barcelona, Spain Geneva, Geneva, Switzerland 6 Transfusional Medicine, Vall d’Hebron Research Institute, 10 Institute of Biological Psychiatry, Mental Health Sct. Hans Hospital, Universitat Autònoma de Barcelona (VHIR-UAB), Barcelona, Spain Roskilde, Denmark 7 Haemostasis and Thrombosis Unit, Department of Hematology, 11 Cardiovascular Medicine Unit, Department of Medicine, Center for Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Thromb Haemost 2022;122:1027–1039. Abstract Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project. Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombo- philia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p ¼ 3.7  106), while the GWAS identified a significant association (rs1687391, p ¼ 3.55  1010) near the ORM1 gene, on chromosome 9. An eQTL Keywords (expression quantitative trait loci) analysis revealed a significant association between ► immunothrombosis the lead GWAS variant and the expression of ORM1 in whole blood (p ¼ 6.14  109). ► neutrophil Additionally, ORM1 expression correlated with levels of cfDNA (p ¼ 4.38  104). extracellular traps Finally, genetic correlation analysis between cfDNA and thrombosis identified a ► α-1-acid glycoprotein suggestive association (ρg ¼ 0.43, p ¼ 0.089). 1 All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, ► expression which might contribute to the susceptibility to thrombosis through mechanisms of quantitative trait loci immunothrombosis. received DOI https://doi.org/ © 2022. The Author(s). March 17, 2021 10.1055/s-0041-1742169. This is an open access article published by Thieme under the terms of the accepted after revision ISSN 0340-6245. Creative Commons Attribution-NonDerivative-NonCommercial-License, October 20, 2021 permitting copying and reproduction so long as the original work is given published online appropriate credit. Contents may not be used for commercial purposes, or March 10, 2022 adapted, remixed, transformed or built upon. (https://creativecommons.org/ licenses/by-nc-nd/4.0/) Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany 1028 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. Introduction eases,6 and atherosclerosis.7 Elevated levels of NETs, through excessive production or defective removal of NETs, may lead Cell-free DNAs (cfDNAs) are extracellular small double- to these clinical conditions.8 Moreover, damaging effects and stranded DNA molecules found circulating in blood. They impaired tissue function may correlate with NET formation.9 have gained importance recently in a wide variety of patholo- NETs have been identified as an important link between gies as a promising disease biomarker.1,2 cfDNA is considered a innate immunity, inflammation, and coagulation, which surrogate marker of neutrophil extracellular traps (NETs), describes the concept of immunothrombosis.10 In vitro and since circulating DNA is its major structural component that in vivo studies have shown that DNA and histones of NETs increases with NET deposition. NETs consist of a complex provide scaffolding for platelet adhesion and promote fibrin extracellular network of nuclear DNA fibers, decorated with deposition in the vasculature by enhancing the activity of histones and antimicrobial proteins that trap and kill patho- blood coagulation factors and promoting thrombosis.11–13 gens to prevent microbial dissemination into the tissue or the Accordingly, cfDNA levels have been found elevated in deep vasculature. The release of DNA into the circulation from vein thrombosis (DVT) both in animals and humans.11,14–16 neutrophils was described in 2004 by Brinkmann et al3 as a Furthermore, in vivo DNase 1 treatment is effective in novel mechanism of defense of the innate immune system to eliminating cfDNA from the blood stream and prevent DVT kill pathogens. This mechanism is called NETosis. It is a unique in mice.16 Also, elevated levels of cfDNA have been able to form of programmed cell death by which active neutrophils predict mortality in venous thrombosis (VT) patients aged extrude their cell material to form NETs. >65 years.17 Most recent findings relate the systemic coa- Recently, NETs have been the focus of intensive investiga- gulopathy and thromboembolic events, frequently observed tion due to their role in innate immunity and its association in patients with severe acute respiratory syndrome-corona- with various diseases, either of infection origin or sterile virus 2 infection, to elevated NET formation.18 Indeed, inflammation, such as sepsis,4 cancer,5 autoimmune dis- patients with coronavirus disease 2019 (COVID-19) have Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. 1029 shown elevated serum levels of cfDNA and other biomarkers Laboratory Measurements of NETs that correlate with disease severity.19 cfDNA of NETs is a biomarker for disease, but may also act Sample Recruitment as a causal pathogenic mediator that contributes to the Thrombophilic participants were not using oral anticoagu- procoagulant phenotype observed in various diseases. lants and antiplatelet drugs at the time of sampling (not used Thus, the identification of the mechanisms that control at least 15 days before the extraction), and were not under DNA release and maintenance into the circulation could heparin treatment (for at least 24 hours before). Blood was help to understand the functional role of cfDNA in physio- obtained at least 3 months after the thrombotic event in logical and pathological conditions. Also, it would help affected individuals and at least 1 month after an acute provide insights for the development of therapies against inflammatory event to minimize the influence of the acute harmful amounts of cfDNA. phase. Blood was collected from the antecubital vein from The mechanisms and genetic factors that predispose to fasting subjects by venipuncture in 1/10 volume of 0.129 elevated levels of cfDNA are largely unknown. To the best of mol/L sodium citrate. After centrifugation at 2,000 g for our knowledge, there is only one study that aimed to identify 25 minutes at room temperature, platelet-poor plasma was the genetic determinants underlying the control of cfDNA obtained and used for the biochemical assays. The remaining levels. In this study, the gene UDP glucuronosyltransferase plasma was stored at 80°C until used. The fraction of blood family 1 member A1 (UGT1A1) on chromosome 2 was identi- cells was washed (1:1) with sodium chloride 0.9% and fied as a regulator of cfDNA levels in serum by a genome- centrifuged for 10 minutes at 1,580 g to obtain the buffy wide association study (GWAS) in a cohort of young adults coat, which was stored at 20°C until DNA extraction. In participating in the Cardiovascular Risk in Young Finns addition, 2.5 mL of blood was collected in a PAXgene Blood Study.20 RNA Tube (QIAGEN GmbH, Germany) and stored at 80°C In our study, we conducted a GWAS to identify genes that until RNA extraction according to the manufacturer’s regulate plasma levels of cfDNA using related individuals of instructions. the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project. In addition, we attempted to identify a candidate DNA Isolation effector transcript by using gene expression data of these Total DNA from the buffy coats of GAIT-2 samples was individuals and elucidate the role of the locus mediating the isolated by a standard salting-out procedure.22 An aliquot GWAS effect. of DNA was stored at 4°C until used for subsequent genotyp- ing of the subjects. The remaining genetic material was kept at 20°C for long-term storage. Methods Population RNA Isolation The GAIT-2 project included 935 individuals from 35 extend- For each individual of the GAIT-2 study, intracellular RNA ed Spanish families selected through a proband with idio- from whole blood collected in PAXgene Blood RNA Tubes was pathic thrombophilia. All of the individuals were recruited at isolated following the protocol of the PAXgene Blood RNA Kit the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) (QIAGEN GmbH, Germany). Total RNA was stored at 80°C from 2006 to 2010. The number of members per family until used. ranged from 11 to 79 (mean  standard deviation [SD]: 33 individuals  17). All of the families included in the project cfDNA Measurement were required to have at least 10 living individuals in three or The cfDNA was quantified in duplicate in plasma from each more generations. Idiopathic thrombophilia was defined as: individual by fluorescence as previously documented by (1) recurrent venous and/or arterial thrombosis (AT; with at Fuchs et al.11,15 In brief, 100 µL of 1/10 diluted plasma in least one spontaneous thrombotic event), (2) a single spon- sterile ultra-pure grade phosphate-buffered saline (PBS; taneous thrombotic event with a first-degree relative also Amrek) was mixed with 100 µL of PBS containing Sytox affected, or (3) early-onset thrombosis (<45 years), with the Green (Life Technologies) at 2 µM final concentration to causes of thrombophilia excluded at inclusion. Accordingly, label DNA. Autofluorescence was background and deter- probands with deficiencies of coagulation proteins or cofac- mined in samples mixed with PBS without Sytox Green. tors including protein S, protein C, antithrombin, plasmino- Samples were incubated for 15 minutes with Sytox Green gen, factor V Leiden, heparin cofactor II, activated protein C in 96-well microplates (FluoroNunc F96 MicroWell plates, resistance, dysfibrinogenemia, lupus anticoagulant, and Nunc) protected from light. After incubation, fluorescence antiphospholipid antibodies were not included. In the was measured at 485 nm excitation and 538 nm emission GAIT-2 study, 118 individuals had been diagnosed with wavelengths in a microplate reader fluorometer (Tecan thrombosis, with a total of 187 thrombotic events (121 Infinite 200Pro). cfDNA concentrations were calculated venous and 66 arterial) registered. Among the individuals through a standard curve of known DNA concentrations with thrombosis, 71 had VT only, 33 had AT only, and 14 had (Human genomic DNA 100 µg/mL, Novagen) and expressed both VT and AT. Detailed composition of the families and the in ng/mL. Measurements of cfDNA were available in 934 collection of lifestyle, medical, and family history data have GAIT-2 subjects. As differences in mean and dispersion of been previously described.21 cfDNA values (ng/mL) were observed among plates, the Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). 1030 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. median value of cfDNA of each plate was subtracted from the ECLIPSE, version 8.1.1),25 which is specifically designed for value of each sample, and divided by the SD of each plate for the genetic analyses in extended pedigrees. normalization. Plate-normalized cfDNA values were used in the analyses in which cfDNA was a covariate, while original Heritability values were used when mixed models including plate as The heritability for cfDNA levels is the estimated proportion random effect were tested. The assessed inter- and intra- of total variance of the cfDNA concentration that is attribut- assay variation coefficients for cfDNA measurement were 6.6 able to the additive effects of genes. It was calculated in GAIT- and 3.4%, respectively. 2 using variance components methods adjusted for covari- ates age, age squared (age2), and sex. Genotyping and Imputation of Samples All of the individuals in the GAIT-2 study were genotyped Genetic and Environmental Correlation of cfDNA with with a combination of two human genotyping arrays from Thrombosis Illumina (San Diego, California, United States): HumanOm- Genetic and environmental correlation coefficients were niExpressExome-8v1.2 (324 individuals and coverage quantified in GAIT-2 between cfDNA concentration and 964,193 variants) and HumanCoreExome-12v1.1 (610 indi- thrombosis (including VT, AT, and both), as well as with VT viduals and coverage 542,585 variants). The variants with a only and AT only. Genetic correlation is of special interest genotype call rate <0.98, a minor allele frequency (MAF) because it quantifies the additive genetic effects that are <0.01, or failing the Hardy-Weinberg equilibrium (HWE) test common to both traits. For that, we tested a bivariate (p < 106) were excluded from the analysis. HWE was calcu- variance component model adjusted for the significant cova- lated by a standard χ2-test with 1 degree of freedom and it riates specific for each trait. Significance was calculated using was tested using parental data only. After filtering the data- the likelihood ratio test (LRT).26 All of the models were sets, we merged the data and obtained 485,672 variants in all corrected for family structure and proband ascertainment the samples. We then imputed genotypes to the 1000 bias. This ascertainment scheme was used to obtain the best Genomes Project phase 1 panel. After imputing, variants estimation for general population parameters. with MAF <0.005 were excluded. After quality control (QC), 10,844,567 variants remained. Imputed variants were then Genome-Wide Association Study mapped along the genome according to the human genome A GWAS with the imputed genotypes was conducted in two assembly GRCh37 (hg19) from the Genome Reference phases in GAIT-2 for transformed values of cfDNA levels to Consortium. identify single-nucleotide polymorphisms (SNPs) regulating interindividual variance of this quantitative trait. First, we RNA Sequencing performed a fast screen of variants with Matrix eQTL27 using The messenger RNA (mRNA) levels of the genes expressed in a linear model, adjusted for age, age2, sex, and including the whole blood were obtained in the GAIT-2 population by RNA kinship matrix to correct for relatedness. Variants with p sequencing (RNA-Seq). In brief, 928 GAIT-2 individuals with <103 were then recalculated in a second phase with the high-quality RNA samples were eligible for RNA-Seq analy- Solarius package24 using the same covariates. All p-values sis. RNA samples were prepared for sequencing with the were calculated by LRT.26 Only the p-values calculated with Illumina TruSeq sample preparation kit (Illumina, San Diego, SOLAR are reported and are represented in a Manhattan plot. California, United States) according to manufacturer’s The genome-wide significance threshold for SNP–phenotype instructions and were sequenced on a HiSeq2000 Sequenc- associations was set at the commonly accepted level of p  ing System (Illumina). The 49-bp sequenced paired-end 5  108. Regional plots of the significant associated loci with reads were mapped to the GRCh37 reference genome with cfDNA levels were generated by LocusZoom.28 the Burrows–Wheeler Aligner v0.5.9.23 We used genes de- fined as protein coding in the GENCODE 10 annotation. We Conditional Association Analysis excluded samples that failed in the library preparation or A second round of SNP–phenotype association analysis for sequence process, those with less than 10 million reads, and cfDNA levels in GAIT-2 was performed by further condition- those with sequences that did not correspond to actual ing on the top associated SNP identified in the GWAS to genotype data. After QC, 16,748 mRNAs from whole blood identify secondary association signals in a particular geno- of 915 GAIT-2 individuals were available for further tran- mic region. scriptome analyses. RNA-Seq data were normalized by the effect of technical covariates, age, sex, and blood cell counts cis-eQTL Analysis for Candidate Genes (by eliminating the variance explained by the first four The identification of SNPs associated with the variability of principal components in the principal component analysis) gene expression levels are known as expression quantitative and then, used in the analyses as residualized values. trait loci (eQTLs), which helps to understand the functional role of the SNP–phenotype associations of a GWAS. cis-eQTL Statistical Analyses variants likely affecting proximal gene expression in whole All analyses were calculated using the R (v.3.6.0; http:// blood were analyzed for the genes of interest in our study www.R-project.org) package Solarius,24 as the interface of (ORM1, ORM2, and COL27A1). For that, linear mixed models the Sequential Oligogenic Linkage Analysis Routines (SOLAR- adjusted for the significant covariates age, age2, sex, and Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. 1031 kinship matrix were conducted in GAIT-2 using FastQTL29 to Table 1 Demographic characteristics of the GAIT-2 subjects evaluate associations between imputed SNPs located within 1 megabase (Mb) upstream or downstream of the corre- Characteristics GAIT-2 sponding gene transcription start site and expression levels No. of subjects, n 935 of the candidate gene transcripts. p-Values were adjusted for No. of families 35 multiple testing by applying the Bonferroni correction. Re- No. of subjects with 118 (12.62) gional plots of the significant eQTLs were generated by thrombosis, n (%) LocusZoom.28 The R package LocusCompareR was used to • VT only, n (%) 71 (7.60) generate plots for visualization of GWAS-eQTL colocalization events. The cis-eQTLs identified in GAIT-2 for candidate • AT only, n (%) 33 (3.52) genes were compared with data from the Genotype-Tissue • Both VT and AT, n (%) 14 (1.50) Expression (GTEx) Portal V8. Mean age, years  SD, range 39.51  21.43, [2.6–101.1] Associations of cfDNA Levels with Gene Expression of Males, n (%) 465 (49.73) Candidate Genes a Current smokers , n (%) 225 (24.06) Linear associations were performed using mixed models be- b tween cfDNA levels and the gene expression in blood of the Oral contraceptives , n (%) 39 (8.30) candidate genes in our study, considering significance at a BMI, mean  SD 24.77  5.58 Benjamini–Hochberg (B–H) false discovery rate (FDR) <0.05. cfDNA (ng/mL), mean  SD 335.97  66.39 This analysis is useful for prioritizing the best candidate genes Heritability of cfDNA  SE (p) 0.264  0.065 to explain associations with circulating cfDNA levels. (p ¼ 3.7  106) Pathway Enrichment Analysis Abbreviations: AT, arterial thrombosis; BMI, body mass index; cfDNA, Biological process and pathway analysis can increase the cell-free DNA; GAIT-2, Genetic Analysis of Idiopathic Thrombophilia; SD, standard deviation; SE, standard error; VT, venous thrombosis. understanding of gene function. Accordingly, the Database Note: Heritability was calculated adjusting for age, age2, and sex. p- for Annotation, Visualization and Integrated Discovery (DA- Value 0.05 was considered statistically significant. VID) v6.8 (https://david.ncifcrf.gov/) is an algorithm useful a Individuals in the study were considered as current smokers when they for discovering enriched functional-related gene groups for smoked, independently of the number of cigarettes. b pathway analysis.30 DAVID annotation tool was used to Oral contraceptives use at inclusion. The percentage of oral contra- ception is expressed with respect to the total number of females extract biological meaning from large gene lists obtained included in the study (N ¼ 470). by our RNA-Seq. GWAS for cfDNA in GAIT-2 Results We conducted a GWAS to identify genetic variants associated Population Description with cfDNA levels in plasma from the subjects of the GAIT-2 Characteristics of the GAIT-2 participants are provided study. Among the >10 million of imputed variants in our in ►Table 1. The mean age of the GAIT-2 population GWAS, we found one significant locus, with rs1687391 as (N ¼ 935) was 39.51 years  21.43 of SD, ranging from the lead SNP (rs1687391-T, β ¼ 0.804, p ¼ 3.55  1010). The youngest individual with 2.6 years to the oldest one with percentage of the variance in cfDNA levels explained by 101.1 years. There were nearly an equal number of males the effect of one copy of the rs1687391-T allele was estimat- (49.73%) and females (50.27%) in the study, with 24.06% of ed in 0.0495%. The SNP rs1687391 is an intergenic variant subjects considered current smokers. located 1.25 kb downstream ORM1 (orosomucoid 1) gene, The covariates that showed a significant effect on cfDNA and 2.06 kb upstream ORM2 (orosomucoid 2) gene on chro- levels in GAIT-2 were age (β ¼  0.015; p ¼ 0.0023), age2 mosome 9q32. A Manhattan plot showing the SNP associa- (β ¼ 0.652; p ¼ 1.75  107), and sex (β ¼ 0.164; p ¼ 0.0074). tion signal with cfDNA levels at the ORM locus is shown Further analyses were adjusted for the effect of these covariates. in ►Fig. 1, along with a regional association plot. A box plot of The heritability for cfDNA in GAIT-2 was 0.26  0.06 of the association of cfDNA levels in the GAIT-2 sample with the standard error (p ¼ 3.7  106) (►Table 1). This indicates that genotype of the lead SNP is given in ►Supplementary Fig. S1 a considerable proportion of cfDNA variance is due to the (available in the online version). The top SNP associations additive effect of genes. with cfDNA levels of the GWAS are shown in ►Table 2. The mean value of cfDNA levels measured in the GAIT-2 Further GWAS data including all SNP–phenotype associa- individuals was 335.97 ng/mL  66.39 of SD (►Table 1). A tions with p < 104 are shown in ►Supplementary Table S2 genetic correlation was found between cfDNA and thrombo- (available in the online version). A conditional analysis was sis (considering cases of VT, AT, and both VT and AT), conducted on chromosome 9 conditioning on the lead SNP although not significant (ρg ¼ 0.43, p ¼ 0.089). Also, a lack rs1687391 at the ORM locus to identify secondary associa- of significant genetic correlation with cfDNA was observed tion signals in this genomic region. The results of this for VT (ρg ¼ 0.41, p ¼ 0.135) or AT (ρg ¼ 0.58, p ¼ 0.186) analysis revealed that no other SNPs were significantly (►Supplementary Table S1 [available in the online version]). associated with cfDNA (►Fig. 1), indicating that Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). 1032 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. Fig. 1 Manhattan plot of the GWAS for cfDNA levels in GAIT-2. (A) Manhattan plot displaying the strength of genetic association (y- axis ¼  log 10(p-value)) of cfDNA levels versus chromosomal position (x-axis ¼ Chromosome). Each dot represents a genotyped or imputed SNP that remained significant after filtering through Matrix eQTL, the second phase of genomic association. The red horizontal line represents the threshold for genome-wide significance (p ¼ 5  10 8) and the blue line indicates the threshold for suggestive associations (p ¼ 10 6). The most genome-wide significant SNP is the rs1687391, which is located on chromosome 9 at the intergenic region between ORM1 and ORM2 loci. (B) Regional plots to depict the independent association signal at the ORM locus. Each dot represents one SNP. Dot colors signify the degree of pairwise correlation (r 2) with the top SNP (purple dot), as presented in the color key. From left to right, LocusZoom of the association signal in the ORM locus for the GWAS; association signal conditioned on the lead SNP (rs1687391). The SNPs (colored dots) significantly associated with cfDNA dropped below significance after conditioning, indicating these SNPs belong to the same association signal. cfDNA, cell-free DNA; eQTL, expression quantitative trait loci; GWAS, genome-wide association study; SNP, single nucleotide polymorphism. rs1687391, which shows the strongest statistical evidence variant for ORM1 expression in our sample was rs1249761 for association, may capture most of the phenotypic varia- (rs1249761-C; β ¼ 0.73; p ¼ 8.17  1065), an intron variant tion at this locus. for COL27A1, which was also an eQTL for ORM2 in GAIT-2 (rs1249761-C; β ¼ 0.53; p ¼ 1.18  1028). The variant was cis-eQTL Mapping for ORM Genes in GAIT-2 also associated with cfDNA levels in the GWAS (rs1249761- To investigate whether ORM1 or ORM2 would be mediating C; β ¼  0.234; p ¼ 7.98  106). The lead eQTL variant for the activity of the significant GWAS locus, cis-eQTLs were ORM2 expression in GAIT-2 was rs1631637 (rs1631637-T; analyzed in the GAIT-2 population for the ORM locus. We β ¼ 0.53; p ¼ 4.36  1031), an intergenic variant located identified 83 cis-eQTL variants for ORM1 expression that 3.71 kb downstream COL27A1 and 8.62 kb upstream ORM1. reached Bonferroni significance (p < 0.05/28,133) This variant was also significantly associated with the ex- (►Supplementary Table S3 [available in the online version]) pression of ORM1 in our sample (rs1631637-T; β ¼ 0.69; and 146 for ORM2 (►Supplementary Table S4 [available in p ¼ 2.42  1063), and showed association with cfDNA levels the online version]). Regional plots showing significant cis- in the GWAS (rs1631637-T; β ¼  0.229; p ¼ 4.97  106). In eQTLs for ORM genes are shown in ►Fig. 2. The lead cis-eQTL addition, the lead SNP rs1687391 in the GWAS for cfDNA Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). Table 2 Top SNP associations with cfDNA levels in GAIT-2 from the GWAS SNP Chr. Position (bp)a Type Closest gene strand Allelesb Minor Allele MAFc βd Vare p-Valuef rs1687391 9 117090006 Intergenic ORM1 þ C/T T 0.057 0.804 0.0495 3.55  1010 rs10982163 9 117091033 Intergenic ORM2 þ G/T/A T 0.059 0.764 0.0473 9.78  1010 rs10982164 9 117091067 Intergenic ORM2 þ G/A A 0.058 0.764 0.0472 9.8  1010 rs112164771 9 117091002 Intergenic ORM2 þ C/T/A T 0.059 0.759 0.0469 1.25  109 rs1687390 9 117089888 Intergenic ORM1 þ G/A A 0.063 0.711 0.0442 3.59  109 rs7040440 9 117091074 Intergenic ORM2 þ C/T/G T 0.068 0.679 0.0427 3.97  109 rs146077223 9 117089921 Intergenic ORM1 þ G/T T 0.063 0.697 0.0425 7.98  109 rs3762056 9 117090434 Intergenic ORM2 þ C/T T 0.063 0.696 0.0425 8.10  109 rs10982154 9 117087212 Intron ORM1 þ T/A A 0.063 0.693 0.0419 9.67  109 rs10982155 9 117087254 Intron ORM1 þ C/T T 0.063 0.693 0.0419 9.68  109 rs145835687 9 117087745 Intron ORM1 þ G/A/T A 0.063 0.692 0.0419 9.84  109 rs10982156 9 117088064 Intron ORM1 þ T/A A 0.063 0.691 0.0418 1.0  108 rs3762055 9 117090575 Intergenic ORM2 þ T/C C 0.063 0.686 0.0416 1.09  108 rs116994374 9 117084672 Promoter ORM1 þ G/A A 0.062 0.693 0.0395 1.73  108 rs113354603 9 117086241 Intron ORM1 þ G/A A 0.066 0.665 0.0391 1.76  108 rs188924106 9 117084722 Promoter ORM1 þ G/A A 0.062 0.689 0.0392 2.06  108 rs150611042 9 117083803 Promoter ORM1 þ C/A/T A 0.063 0.679 0.0379 3.07  108 rs140041983 9 117084971 Promoter ORM1 þ C/G/T G 0.054 0.700 0.0357 1.16  107 rs10921528 1 194259458 Intergenic CDC73 þ G/C C 0.387 0.260 0.0352 3.07  107 rs115078510 4 185917640 Intergenic HELT þ C/T T 0.020 0.995 0.0334 4.30  107 rs1325429 1 194262018 Intergenic CDC73 þ C/T T 0.405 0.248 0.0320 8.36  107 Thrombosis and Haemostasis rs60345835 3 196688877 Intron PIGZ  C/A A 0.315 0.250 0.0291 9.58  107 rs7301073 12 25262868 Intron CASC1  T/C C 0.018 0.976 0.0309 9.73  107 Vol. 122 rs10519385 4 137249906 Intergenic PCDH18  C/T T 0.077 0.437 0.0303 1.00  106 Abbreviations: CASC1: cancer susceptibility candidate 1; CDC73: cell division cycle 73; Chr, chromosome; HELT: helt bHLH transcription factor; ; ORM1: orosomucoid 1; ORM2: orosomucoid 2; PIGZ: phosphatidylinositol glycan anchor biosynthesis, class Z; PCDH18: protocadherin 18. a No. 6/2022 All chromosome positions were based on the human GRCh37/hg19 assembly. b GWAS and Gene Expression for Plasma Levels of cfDNA Alleles aligned to þ strand. Underlined alleles are very rare alleles in some populations, with minor allele frequency (MAF) < 0.01, not present in our sample. c Minor allele frequency of the SNP in our sample. d Effect size of one copy of the minor allele on cfDNA levels. e Percentage of the variance in cfDNA levels that is explained by the effect of one copy of the minor allele. f p-Value of the association with cfDNA levels. Genome-wide significant threshold was set at p  5  108. Most significant SNP–phenotype associations, with p-value  106, are shown in the table. © 2022. The Author(s). Lopez et al. 1033 1034 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. Fig. 2 Regional plots of cis-eQTLs for ORM1 and ORM2 genes. Regional association plots displaying the genomic context of the cis-eQTL signal for ORM1 (A) and ORM2 (B). Each dot represents a genotyped or imputed SNP. Dot colors signify the degree of pairwise correlation (r2 ) with the top SNP (purple dot), as presented in the color key. eQTL, expression quantitative trait loci; SNP, single nucleotide polymorphism. levels was also a significant cis-eQTL variant for ORM1 of the genotypes for the most significant cis-eQTL variants in (rs1687391-T; β ¼ -0.673; p ¼ 6.14  109) and ORM2 ex- whole blood for both genes. pression (rs1687391-T; β ¼  0.691; p ¼ 2.07  108). This The significant cis-eQTL variants identified in our study indicates that ORM1 and ORM2 are the main candidate genes associated with ORM1 and ORM2 expression mostly were that explain the association of cfDNA with genetic variation. regulatory variants of the transcript levels of the two genes. Box plots in ►Supplementary Fig. S2 (online only) illustrate In this regard, 70 variants that reside mainly close to or the expression levels of ORM1 and ORM2 genes as a function within COL27A1 (54%), ORM1 (27%), and ORM2 (17%) Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. 1035 Fig. 3 LocusCompare plots of cis-eQTLs for ORM1 and ORM2 genes. Visualization plots displaying colocalization of GWAS and cis-eQTLs for ORM1 (A) and ORM2 (B) in the GAIT-2 study. Only cis-eQTL variants reaching the Bonferroni threshold of significance (0.05/28,133) are represented in LocusZoom plots showing eQTLs. The labeled SNP (purple dot) is the lead SNP in the GWAS that is a significant eQTL variant for both ORM1 and ORM2 genes. Other SNPs are colored according to their LD r2 with the lead SNP, as presented in the color key. eQTL, expression quantitative trait loci; GWAS, genome-wide association study; LD, linkage disequilibrium; SNP, single nucleotide polymorphism. (►Supplementary Table S5 [available in the online version]) significant regulatory SNPs identified in GAIT-2. Thirty-one were cis-eQTL variants shared by the two genes. Of note, of them were cis-eQTL variants overlapping with our GWAS although more than half of the regulatory variants for ORM1 signal, of which 14 were genome-wide significant SNPs. and ORM2 lay close to or within COL27A1, also being the most significant, none of them were regulatory variants of cfDNA and mRNA Levels of Candidate Genes COL27A1 in whole blood (according to our data in GAIT-2— Finally, associations between cfDNA levels and mRNA levels ►Supplementary Table S6 [available in the online version]— for the candidate genes from the GWAS were calculated to and in agreement to GTEx), therefore the gene COL27A1 is not identify a potential effector transcript and elucidate the role considered a mediator of our GWAS signal. Noteworthy, of the locus mediating the GWAS effect. The expression levels these cis-eQTL variants in common for the two ORM genes of ORM1 in whole blood showed an inverse significant were completely overlapping with the variants associated correlation with cfDNA levels at a FDR < 0.05 (β ¼  0.098; with cfDNA levels. This indicates that cis-eQTLs for ORM p ¼ 4.38  104; FDR ¼ 0.026). This indicates that ORM1 ex- genes capture 100% of the GWAS signal. The overlapping cis- pression decreased with the increase of cfDNA in plasma. eQTL variants are listed in ►Supplementary Table S7 (avail- However, no significant correlation was observed with ORM2 able in the online version). Colocalization of the GWAS signal (β ¼  0.071; p ¼ 0.0245; FDR ¼ 0.199). This supports ORM1 and eQTLs were illustrated through LocusCompare (►Fig. 3). as the main candidate gene in the control of the levels of cfDNA in plasma. Since most significant eQTL variants for cis-eQTLs for ORM Genes in GTEx ORM1 and ORM2 genes were SNPs located close to or within As genotype and expression data in multiple tissues are avail- COL27A1 gene, we tested also its correlation with cfDNA. able in GTEx, we conducted a search for cis-eQTLs for ORM genes However, it was not statistically significant (β ¼  0.069; in GTEx in whole blood and compared with our results. Lead cis- p ¼ 0.0275; FDR ¼ 0.211). These data are shown in ►Table 3. eQTL variants for ORM1 (rs1249761) and ORM2 (rs1631637) that we identified in whole blood were also eQTLs in GTEx for Pathway Enrichment Analysis these genes in the same tissue. The lead variant rs1687391 was To identify functionally related genes from the large list of not reported in GTEx; however, the rs10982154 (an intron mRNA transcripts that correlated with cfDNA in our study, variant of ORM1) was used as a proxy SNP (LD r2 ¼ 1) and and delineate a potential biological pathway for our studied showed association only with ORM1 (p ¼ 1.8  105) and ORM2 phenotype, we conducted a functional enrichment analysis (p ¼ 1.0  108) in whole blood in GTEx. by using the DAVID annotation tool. Accordingly, we per- A total of 79 cis-eQTL variants for ORM1 in whole blood were formed the gene set enrichment analysis with all the tran- reported in GTEx, of which 46 (58.2%) were significant regula- scripts filtered by FDR <0.1 in our RNA-Seq. Thus, a total of tory SNPs identified in GAIT-2. Twenty-five of them were cis- 983 mRNAs showed significant correlation with cfDNA lev- eQTL variants overlapping with the associated locus with els. All of these transcripts were considered for the enrich- cfDNA levels in our GWAS, of which 10 were genome-wide ment analysis and were uploaded into DAVID. A total of 52 significant variants. For ORM2, 103 cis-eQTL variants in whole transcripts (5.29%) were not recognized by the enrichment blood were reported in GTEx, of which 80 (77.7%) were tool, as they mainly were long intergenic noncoding RNAs Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). 1036 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. Table 3 Associations between mRNA levels of candidate genes and plasma levels of cfDNA in GAIT-2 Gene Ensembl Gene ID Chr. position (bp)a βb p-Valuec FDR 5% 4 ORM1 ENSG00000229314 9:117085336–117088755 0.098 4.38  10 0.026 ORM2 ENSG00000228278 9:117092149–117095532 0.071 0.0245 0.199 COL27A1 ENSG00000196739 9:116917840–117074791 0.069 0.0275 0.211 Abbreviations: Chr.: chromosome; COL27A1: collagen type XXVII α 1; ORM1: orosomucoid 1; ORM2: orosomucoid 2. a All chromosome positions were based on the human GRCh37/hg19 assembly. b Effect size of one copy of the minor allele on gene expression levels. c p-Value of the association of gene expression with cfDNA levels. Significance was considered at a false discovery rate (FDR) < 0.05. (lincRNAs) or genes coding uncharacterized proteins. Then, does may exist. The gene UGT1A1 on chromosome 2 was 931 transcripts were finally analyzed by the Functional identified recently by GWAS as a regulator of cfDNA levels Annotation Clustering Tool according to the following func- in serum.20 However, we did not find significant or suggestive tional annotation categories: Gene Ontology (GO) Biological SNP associations at this locus. Several nongenetic factors that Process, GO Molecular Function, GO Cellular Component, influence the phenotype variance, and thus might contribute KEGG pathway, and UniProt Keywords. The results show that to different findings, have been controlled in the analyses of our gene set is mainly enriched in pathways related to the both studies, although others may remain uncontrolled innate immunity, type I interferon (IFN) signaling pathway, (►Supplementary Material [available in the online version]). defense response to virus, and cell division. ►Supplementary Some of them may be related to technical issues (different Table S8 (available in the online version) shows the func- fluorochrome and assay conditions) or differences in popula- tional annotation clusters that have significant group enrich- tions (e.g., case–control vs. family-based, different demo- ment scores, showing the genes associated with each graphic and clinical characteristics). On the other hand, the enriched biology term. higher sample size of the study of Jylhävä et al could contribute Finally, a summary of our study design indicating our with more statistical power to the GWAS study. In addition, most relevant findings for cfDNA in GAIT-2 is provided in both studies might have different pools of cfDNA in plasma or ►Supplementary Fig. S3 (available in the online version). serum (from different origins or cell sources) thus showing different regulatory mechanisms. Furthermore, our study of families with hereditary idiopathic thrombophilia could be Discussion genetically enriched in a specific pathway for cfDNA regulation The potential of plasma levels of cfDNA as a biomarker of (e.g., immunothrombosis) and thus, it could show different disease and pathogenic mediator is currently being investigat- locus than those observed in the study with healthy young ed. However, the factors that regulate cfDNA levels in physio- individuals. In this regard, studies of extended pedigrees are logical and pathological conditions are poorly understood. Our likely to represent a more homogeneous and limited set of aim was to identify genes that control the variation of cfDNA in causative genes and pathways. families with idiopathic thrombophilia from the GAIT-2 study In GWAS studies, the identification of genes mediating the (►Supplementary Material [available in the online version]). activity of SNPs with functional effect is usually a challenging For this purpose, we performed an integrated genomic and task. In this regard, the analysis of eQTLs in disease-relevant transcriptomic data analysis. Our results show that cfDNA is a tissues is useful to detect associations of genetic variation heritable trait, with 26% of cfDNA variance in plasma under with changes in gene expression that may help to understand genetic control. To the best of our knowledge, this is the first the functional role of the SNP–phenotype associations.31 time that the heritability of cfDNA has been reported. It is Accordingly, cis-eQTLs, which have a large effect size on important to note that cfDNA levels show an important genetic the expression of nearby genes, were mapped in GAIT-2 correlation with thrombosis, although it did not reach statisti- for the ORM locus and lead to the identification of the cal significance mainly due to limited sample size, since only variants that likely affect proximal ORM1 and ORM2 expres- the 12.62% of the total GAIT-2 population had thrombosis. This sion in whole blood. Among the number of cis-eQTL variants suggests the presence of common genetic pathways underly- identified in our study for ORM genes, the lead variant ing both phenotypes. rs1687391 in our GWAS was significantly associated with The GWAS shows that a SNP near the ORM1 gene influences both ORM1 and ORM2 expression. This indicates that the the plasma levels of cfDNA, explaining nearly 0.05% of its rs1687391 might be a regulatory variant that captures part of variance (rs1687391-T). The lead SNP showed no association the variability of the expression of ORM genes and, thus it with thrombosis in the whole GAIT-2 population, maybe due could influence cfDNA levels. to limited sample size of cases with thrombosis in our study as We found that most cis-eQTL variants for ORM1 and ORM2 mentioned above (data not shown). The conditional analysis in blood are regulatory SNPs shared by the two genes that on the lead SNP shows that it is an independent GWAS signal. reside mainly close to or within COL27A1 and ORM genes. This indicates that a causal variant within this signal or in Among these cis-eQTL variants, the most significant SNPs strong LD (linkage disequilibrium) with the associated locus modulating gene expression of the ORM locus lie close to or Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. 1037 within COL27A1 (located 10.5 kb upstream ORM1); however, activities.43 AGP is considered a natural anti-inflammatory none of them were eQTL variants for COL27A1. Of note, all the and immunoregulatory protein, with proangiogenic and cis-eQTL variants shared by ORM genes were shared with the antiapoptotic properties that could be important for cardio- GWAS signal, thus providing more evidence of the functional vascular disease.44,45 Accordingly, some evidence supports a role of the ORM locus in the control of cfDNA levels. In role of AGP in coagulation. As mentioned above, AGP has addition, most of the SNPs of the GWAS signal lie in regula- been associated with FX,33,37 thrombin generation poten- tory regions (noncoding and promoter) of ORM1 expression, tial,39 and protein S levels.40 Also, AGP interacts with plas- which suggests ORM1 as the main candidate gene in the minogen activator inhibitor 1, enhancing fibrinolysis control of cfDNA levels. These results are reinforced by the inhibition.46 In an experimental model of septic peritonitis integration of expression data from the main candidate in rats, AGP administration suggests a protective effect of genes, which reveals that among the genes within the AGP against a procoagulant phenotype by normalizing some associated locus, only ORM1 expression correlates negatively clot parameters.47 This is in line with the protective effect of with plasma levels of cfDNA. Accordingly, we hypothesize ORM1 that we suggest. Consistent with our results, Liu et al that ORM1 could have a protective role against harmful reported that AGP reduced in vitro the formation of DNA- effects of elevated cfDNA. While functional assays will be based NETs for sperm entanglement. The study showed that necessary to disclose the causal SNP, our study has provided AGP reduced the phagocytic activity of neutrophils in a dose- strong evidence for a novel gene that regulates cfDNA levels. dependent manner, reducing NET formation and superoxide Furthermore, our RNA-Seq data indicate that the gene set generation of activated neutrophils.48 Also, elevated AGP in associated with cfDNA is mainly related to pathways of body fluids and altered patterns of glycosylation have been innate immunity, type I IFN signaling, defense response to associated with several diseases.49 In particular, elevated virus, and cell division. Type I IFNs have diverse effects on gene expression and plasma levels of AGP have been associ- innate and adaptive immune cells during infection, are ated with coronary disease and stroke.50,51 important for host defense against viruses, and enhance Some limitations in our study should be considered. First, NETosis. IFN-α primes mature neutrophils to form NETs to the measurement of ORM1 protein in plasma is not available bind to pathogens.32 This points to immunothrombosis as a in the GAIT-2 sample. Plasma levels of ORM1 protein and its mechanism by which ORM1 could contribute to the suscep- correlation with the lead SNP, mRNA ORM1 levels or cfDNA, tibility of thrombosis. could have added an extra piece of support to our study. Various genetic variants close to or within ORM1, which Moreover, the low number of cases with thrombosis in have been associated with cfDNA levels in our GWAS, have relation to the whole participants in the family-based study been found associated with traits related to coagulation or may have limited the association of cfDNA or ORM1 variants cardiovascular disease in the NHGRI-EBI GWAS Catalog. For with thrombosis risk. Accordingly, a replication cohort with example, the lead variant rs1687391 has been associated cases and controls is essential. Also, a modest coefficient of with the serine protease inhibitor Kazal-type 2 (SPINK2),33 a variance is attributable to the lead SNP that could be in part protein associated with stroke,34 mean platelet volume and explained by the complex nature of the plasma cfDNA. platelet count,35 and leukocyte count.36 The intergenic However, while not clinically meaningful for prediction of rs3762056 and the intron variant rs10982156 have been the risk of the disease, our finding of a genome-wide signifi- associated with the plasma levels of the coagulation factor X cant signal in the ORM1 gene clearly points to a novel (FX).33,37 The rs1687390 has been associated with variance regulatory mechanism of cfDNA. On the other hand, although in warfarin dose,38 and the rs150611042 in the promoter of direct fluorescent Sytox Green staining is not a validated ORM1 has been reported to influence the interindividual method for the quantification of cfDNA in plasma, it is capacity to generate thrombin.39 Also, SNPs at ORM1 gene extensively used and might serve as reliable alternative to (rs150611042) were recently found associated with protein S real-time quantitative polymerase chain reaction for routine levels through a meta-analysis using GWAS data from the quantification of cfDNA and multicenter standardization.52 CHARGE Consortium.40 These data, along with our results, Also, it has shown higher sensitivity for DNA in plasma than suggest that ORM1 is a susceptibility locus in thrombosis. other intercalating fluorochromes.17 Furthermore, diverse Also, it might be linked to cfDNA-mediated inflammation as forms of DNA may circulate in plasma derived from NETs or an underlying pathological pathway. The ORM locus con- mitochondria, but may also be actively released from living forms a complex and very polymorphic genomic region with cells either encapsulated in vesicles or in the form of com- two highly homologous genes, ORM1 and ORM2, that are plexes, triggering different biological mechanisms associated differentially regulated.41 ORM1 is located 3.55 kb upstream with health and disease. However, fluorescence quantifica- ORM2, and code for α-1-acid glycoprotein 1 (AGP1) and tion of cfDNA is an unspecific approach that measures the AGP2, respectively. Both proteins are present in plasma, total amount of extracellular DNA, and thus it may not be and AGP1 is the most abundant, where it functions as considered a single biomarker of disease. transport proteins. AGP1 is an acute phase protein42 mainly In summary, to the best of our knowledge, we have expressed in liver and secreted in the blood, although it can presented the first integrated study that links genetic varia- be produced also in extrahepatic tissues.43 The exact biolog- tion with gene expression and the levels of cfDNA in plasma ical function of AGP (also known as orosomucoid) is un- in a study of extended families affected with idiopathic known, although it has been related to multiple modulating thrombophilia. Our results should shed light on the genetic Thrombosis and Haemostasis Vol. 122 No. 6/2022 © 2022. The Author(s). 1038 GWAS and Gene Expression for Plasma Levels of cfDNA Lopez et al. control of cfDNA levels, which is considered a risk factor for References VT and suggests that ORM1 is a novel locus regulating cfDNA 1 Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ. The levels in plasma. 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