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A Genetic Variant in the BCL2 Gene Associates with Adalimumab Response in Hidradenitis Suppurativa Clinical Trials and Regulates Expression of BCL2

Open ArchivePublished:August 26, 2019DOI:https://doi.org/10.1016/j.jid.2019.06.152
      Hidradenitis suppurativa is a chronic skin disease with a significant genetic component and prevalence from 0.5% to 4%. Adalimumab is the only treatment approved by either the European Medicines Agency or the U.S. Food and Drug Administration for the management of moderate to severe hidradenitis suppurativa. To identify genetic variants associated with adalimumab response, we performed a genome-wide association study (GWAS) from the most extensive two phase 3 hidradenitis suppurativa clinical trials (PIONEER I and II) to date. Through direct genotyping and imputation, we tested almost 7 million genetic variants with minor allele frequency > 5% and identified one single linkage disequilibrium block, located in the intron of the BCL2 gene, which reached genome-wide significance (lead single-nucleotide polymorphism, rs59532114; P = 2.35E–08). Bioinformatic analysis and functional genomics experiments suggested a correlation of the most strongly associated single-nucleotide polymorphism minor allele with increased BCL2 gene and protein expressions in hair follicle tissues. In reciprocal knockdown experiments, we found that BCL2 is down-regulated by TNF inhibition. These results highlight a pathway that involves BCL2 in response to adalimumab. Further work is required to determine how this pathway influences adalimumab effectiveness in patients with hidradenitis suppurativa.

      Abbreviations:

      AN (abscess and inflammatory nodule), AN.COUNTFC (fold change of the abscess and inflammatory nodule count), eQTL (expression quantitative trait loci), GWAS (genome-wide association study), HiSCR (Hidradenitis Suppurative Clinical Response), HS (hidradenitis suppurativa), LCL (lymphoblastoid cell line), MAF (minor allele frequency), SNP (single-nucleotide polymorphism), TNF (tumor necrosis factor)

      Introduction

      Hidradenitis suppurativa (HS, also designated acne inversa) is a chronic skin disease characterized by painful, deep-seated, and recurrent inflamed subcutaneous nodules. Follicular occlusion is the primary event resulting in comedones, painful nodules, abscesses, and recurrent draining sinuses in the apocrine gland–bearing skin ultimately culminating in disfiguring scars (
      • De Vita V.
      • McGonagle D.
      Hidradenitis suppurativa as an autoinflammatory keratinization disease.
      ,
      • Saunte D.M.L.
      • Jemec G.B.E.
      Hidradenitis suppurativa: advances in diagnosis and treatment.
      ). The prevalence of HS is 0.5–4% but maybe underestimated (
      • Garg A.
      • Lavian J.
      • Lin G.
      • Strunk A.
      • Alloo A.
      Incidence of hidradenitis suppurativa in the United States: a sex- and age-adjusted population analysis.
      ,
      • Revuz J.E.
      • Canoui-Poitrine F.
      • Wolkenstein P.
      • Viallette C.
      • Gabison G.
      • Pouget F.
      • et al.
      Prevalence and factors associated with hidradenitis suppurativa: results from two case-control studies.
      ). Acquired and constitutive risk factors associated with HS include smoking, obesity, infection, and family history. Familial aggregation of HS suggests that genetic factors play an important role in disease pathogenesis. For example, up to 42% of patients report a family history of HS, and in some families, HS can follow an autosomal dominant inheritance mode (
      • Von der Werth J.M.
      • Williams H.C.
      • Raeburn J.A.
      The clinical genetics of hidradenitis suppurativa revisited.
      ,
      • Fitzsimmons J.S.
      • Guilbert P.R.
      A family study of hidradenitis suppurativa.
      ). Two large multigeneration pedigrees found 100% penetrance as an autosomal dominant trait (
      • Al-Ali F.M.
      • Ratnamala U.
      • Mehta T.Y.
      • Naveed M.
      • Al-Ali M.T.
      • Al-Khaja N.
      • et al.
      Hidradenitis suppurativa (or acne inversa) with autosomal dominant inheritance is not linked to chromosome 1p21.1-1q25.3 region.
      ). For most patients, HS appears to be a complex trait with limited knowledge about the pathogenesis.
      Adalimumab is the only approved treatment for the management of moderate to severe HS by either the European Medicines Agency or the U.S. Food and Drug Administration, with an overall response rate approaching 60% (42% and 59% for PIONEER I and PIONEER II respectively;
      • Kimball A.B.
      • Okun M.M.
      • Williams D.A.
      • Gottlieb A.B.
      • Papp K.A.
      • Zouboulis C.C.
      • et al.
      Two phase 3 trials of adalimumab for hidradenitis suppurativa.
      ). In addition to adalimumab, conventional therapies for HS include surgical excision of affected sites, topical antibacterial agents, antiseptic cleansers, intralesional corticosteroids, systemic antibiotics, systemic corticosteroids, or hormonal therapies, which result in varied and often only temporary improvement (
      • Saunte D.M.L.
      • Jemec G.B.E.
      Hidradenitis suppurativa: advances in diagnosis and treatment.
      ). Given the limited knowledge of the pathways involved in HS etiology or the differential HS patient response to anti–tumor necrosis factor (TNF) treatment, we performed an unbiased GWAS to identify variants associated with adalimumab response.
      This analysis resulted in a single genome-wide significant hit, which was followed up with functional experiments to determine which gene(s) in the local linkage disequilibrium block, if any, is directly affected by the genetic variant. These functional experiments were performed in HS-relevant primary cell types to illuminate the tissues/cell types that are potentially relevant to the mechanism through which this variant influences adalimumab response in patients with HS.

      Results

      Adalimumab response GWAS of HS

      All participants of both studies who received adalimumab either in the initial treatment period 1 or in periods 2 or 3 were combined to increase the statistical power (Figure 1). A GWAS against the log2 fold change of the abscess and inflammatory nodule (AN) count (AN.COUNTFC) was performed, yielding a single peak with a significant association. Five single-nucleotide polymorphisms (SNPs) on chromosome 18 within this genome-wide analysis significantly associated with adalimumab response and had an effect size estimate of approximately 1.07 (± 0.37) units of log2 AN.COUNTFC per minor allele (Table 1). All five SNPs were located in the second intron of the BCL2 gene. These variant SNP genotypes have minor allele frequencies (MAFs) of 0.04 in the Caucasian population in the 1000 Genomes database. However, they are relatively common in African American (MAF = 0.27) and Asian (MAF = 0.12) populations.
      Figure thumbnail gr1
      Figure 1GWAS study design for HS response to adalimumab. (a) Subjects in red boxes were included to maximize the statistical power. Ancestry outliers: subjects from European cluster with >1.9 times the estimated standard deviation of Utah residents with Northern and Western European ancestry from the Centre d'Etude du Polymorphisme Humaine collection reference cluster in the 1000 Genomes Reference Panel. (b) Overall decay of Log2 AN.COUNTFC. Red line represents Log2 AN.COUNTFC after treatment (4, 8, and 12 weeks) compared with baseline (0 weeks). Change above the red line represents symptoms worsening, whereas change below the redline represents symptoms improved. Each circle represents one patient. (c) Distribution of AN.COUNTFC at week 12. On average with each treatment time point, the percentage of AN.COUNT decreases to 50% of the original level; all the y-xiles of AN.COUNTFC was Log2 transformed. AN.COUNT, abscess and inflammatory nodule count; EW, every week; GWAS, genome-wide association studies; HS, hidradenitis suppurativa; PBO, placebo; QC, quality control.
      Table 1SNPs in the Chr18 Loci Associated with HS to Adalimumab Response in European Ancestry
      ChrrsIDPosition
      SNP position, build 37.
      GenesAllele
      Major/minor allele.
      EAFP-Value
      chr18rs5953211460830032BCL2C/A0.042.35E–08
      chr18rs6764577860836331BCL2C/T0.043.65E–08
      chr18rs1187747560831189BCL2T/C0.044.26E–08
      chr18rs1187791160841744BCL2C/G0.044.30E–08
      chr18rs1115237060835509BCL2G/C0.044.62E–08
      chr18rs1707079860840128BCL2A/G0.045.43E–08
      chr18rs6040510760405107BCL2C/G0.041.51E–07
      chr18rs14105751660836654BCL2C/T0.012.38E–07
      chr18rs14509965160836658BCL2C/T0.022.38E–07
      Abbreviations: Chr, chromosome; EAF, effect allele frequency in Caucasian; HS, hidradenitis suppurativa; SNP, single-nucleotide polymorphism.
      1 SNP position, build 37.
      2 Major/minor allele.

      Replicating the association in other sample sources

      Because the two clinical trials (PIONEER I and II) are the only HS trials with adalimumab treatment for which we had access, we pursued replication in two other sources of data, although they are not all completely independent.
      In the first data source (Data Source 1), we tested additional time points (week 4 and week 8) that were not used in the original GWAS (GWAS identified association with a response at week 12, which was the primary outcome time point; Figure 2c). As in the week 12 data, in both week 4 and week 8, there were significant associations between the SNP allele status and the AN.COUNTFC (P = 0.0003 and P = 0.037, respectively; Figure 2c left panel).
      Figure thumbnail gr2
      Figure 2Overview of adalimumab response GWAS results in HS. (a) Manhattan plot for a null hypothesis of no additive association to genotype was tested for each variant and –log10 P-value is given. (b) The zoomed plot surrounding the genome-wide significant SNPs on chromosome 18 in the BCL2 gene. Top SNP rs59532114 in purple, and linkage disequilibrium SNPs with r2 > 0.8 in red; r2 > 0.6 in orange; r2 > 0.4 in green. (c) Additional data sources support the association analysis. Data source 1 are subjects at different time points (weeks 4, 8, and 12) (P-value of 0.0003, 0.037, and <5E–08, respectively). Data source 2 are subjects but in 24 weeks washout and return to adalimumab treatment and significant difference in week 12 (P = 0.00017). The light blue box represents patients homozygous for the major allele (CC), whereas the dark blue box represents patients heterozygous of the minor allele (CA). GWAS, genome-wide association study; LD, linkage disequilibrium; SNP, single-nucleotide polymorphism.
      In a second data source (Data Source 2), we compared the response of the same individuals, but in the open-label extension trial that followed the initial trials after a 12-week washout period (Figure 1). All the participants enrolled in period 3 experienced a 12-week washout period, following which they were retreated with adalimumab 40 mg every week (EW) for up to 48 weeks. Within period 3, we tested the top GWAS variant for association with the response in each of the five time points after returning to adalimumab dosing. We found a significant association between the rs59532114 genotype and response (P = 0.00017 at week 12 and P = 0.038 at week 72). However, in this postwashout time course, there was no significant association in weeks 4 and 8 (P = 0.14 and P = 0.32, respectively; Figure 2c, right panel). All measured time points in the postwashout time course had effect size estimates in the same direction as the original GWAS.

      rs59532114 association with change in AN count is drug dependent

      To ask if the effect of this SNP was drug dependent, we compared individuals treated with adalimumab to those who never participated in a treatment arm. As shown in the box plots in Figure 3, within the adalimumab-treated group, the participants who carried the minor allele had inadequate response compared with other participants. In contrast to the general pattern of decreasing AN count after 12 weeks of adalimumab treatment, the average AN.COUNTFC in the minor allele group was >1, which indicated that participants in this subgroup had increased AN count compared with their baseline (Figure 3, left box). In contrast, within the placebo group, there was no difference between reference allele carriers and minor allele carriers (P = 0.64; Figure 3, right box), suggesting the association is dependent on adalimumab treatment. Analysis between the SNP rs59532114 genotype with Hurley stages did not show any association (Supplementary Figure S1).
      Figure thumbnail gr3
      Figure 3The box plot shows SNP the minor allele at rs59532114 associated with increased AN.COUNTFC and less response at week 12 in HS trials in patients treated with adalimumab (P < 0.001). Each dot represents the AN.COUNTFC of a participant. In the placebo group, no significant difference between the major and minor allele of rs59532114 (P = 0.64). The horizontal lines within each box represent the lower and upper borders of each box with 25th and 75th percentiles, respectively, and the whiskers mark the 95% confidence intervals. AN.COUNTFC, fold change of the abscess and inflammatory nodule count; HS, hidradenitis suppurativa; SNP, single-nucleotide polymorphism; WT, patients homozygous for the major allele (genotype CC); W/V, patients heterozygous for the minor allele (genotype CA).

      Expression quantitative trait loci analysis

      Because the significantly associated variants are all within the second intron of BCL2, we sought to test whether they affected this gene or other neighborhood genes using analysis of expression quantitative trait loci (eQTL). We evaluated the top two equally linked SNPs, rs59532114 and rs67645778 (linkage disequilibrium, R2 = 1), for their association with expression levels of genes (BCL2, PHLPP1, KDSR, VPS4B, and SERPINB5) within a 1-Mb region using published eQTL data (Figure 2b), such as the Genotype-Tissue Expression project.13 However, with an allele frequency of 0.04 in the Caucasian population, there were few minor allele genotype samples in the Genotype-Tissue Expression database, so we expanded our analysis to another public dataset (GSE24277), which includes 300 lymphoblastoid cell lines (LCLs) from three ethnic groups. We found that rs59532114 was an eQTL for BCL2 in African Americans and Han Chinese Americans participants (Supplementary Figure S2).
      We also identified a recently published whole-genome sequencing study of LCLs from UK10K (Rare Genetic Variants in Health and Disease project) participants, which covered rare variants and more LCLs compared with Genotype-Tissue Expression (N = 506). In this study, the SNP rs67645778, which is in equal linkage disequilibrium with rs59532114, was in eQTL with BCL2 with P = 9.45E–07 (
      • Brown A.A.
      • Viñuela A.
      • Delaneau O.
      • Spector T.D.
      • Small K.S.
      • Dermitzakis E.T.
      Predicting causal variants affecting expression by using whole-genome sequencing and RNA-seq from multiple human tissues.
      ). Finally, to confirm that rs59532114 is an eQTL for BCL2, we designed a more balanced study concerning genotype. We selected 72 LCLs from Caucasians in the 1000 Genomes Project human variation repository panel with known genotypes at rs59532114 that were either homozygous for major allele participants (n = 18), heterozygous (n = 34), or homozygous for the minor allele (n = 18). We cultured these LCLs for 72 hours for mRNA extraction to test the SNP correlation to expression of BCL2 measured by quantitative PCR. We found higher BCL2 mRNA expression in LCLs homozygous for the minor allele genotype as compared with LCLs homozygous for the major genotype (P = 5.75E–04; Figure 4a ). Other genes in the region, including PHLPP1, KDSR, VPS4B, and SERPINB5, were not significantly associated with allele status (data not shown).
      Figure thumbnail gr4
      Figure 4The minor allele of rs59532114 genotype-carried LCLs associates with increased BCL2 mRNA and protein levels. (a) QRT-–PCR of BCL2 expression based on different genotype classes of LCLs (CC, n = 18; CA, n = 36; AA, n = 18). The homozygote genotype of minor allele (AA) genotype was associated with increased mRNA expression of BCL2. (b) Western blot and (c) quantification of BCL2 protein (28 kDa) were more highly expressed in the minor allele homozygote LCLs compared with major allele homozygote LCLs. ACTB (42 kDa) was used as a control (upper). (d) No differences between minor allele homozygote LCLs to major allele homozygote LCLs for other proteins (BCL2, ACTB, PHLPP1, KDSR, VPS4B, and SERPINB5) locate with 1 Mb around the SNP. LCL, lymphoblastoid cell line; QRT-PCR, quantitative real-time reverse transcriptase–PCR; SNP, single-nucleotide polymorphism.

      SNP rs59532114 minor allele associated with increased baseline BCL2 protein level in LCLs

      In the LCLs, the SNP rs59532114 minor allele was associated with increased BCL2 mRNA expression, but only a fraction of eQTL translates to functional protein differences (
      • Battle A.
      • Khan Z.
      • Wang S.H.
      • Mitrano A.
      • Ford M.J.
      • Pritchard J.K.
      • et al.
      Genomic variation. Impact of regulatory variation from RNA to protein.
      ). We tested if the minor allele also associated with BCL2 protein levels. We randomly selected four LCLs homozygous for the major allele and four LCLs homozygous for the minor allele from the same LCLs that were used for previous eQTL analysis. Cytoplasmic proteins were extracted for western blot analysis. As shown in the gel image, all the homozygous major allele LCLs have less BCL2 protein compared with the LCLs homozygous for the minor allele (28 KDa band; Figure 4b and c). As in the eQTL analysis, we did not find expression correlation with the protein levels of the neighborhood proteins (Figure 2b) such as PHLPP1, VPS4B, KDSR, and SERPINB5 (Figure 4d).

      rs59532114 minor allele associated with increased BCL2 transcriptional activity in reporter assays

      To evaluate the effect of SNP rs59532114 on BCL2 transcriptional activity in HS disease–relevant tissues, we created luciferase reporter constructs containing 250 bp of DNA surrounding this top SNP. We cloned the full length of the 2.8-kb BCL2 promoter downstream of the SNP region (Figure 5a and b) and performed the reporter assay in three different potentially relevant tissues for HS. To compare expression between the major and minor allele, we generated two reporter constructs with only one single nucleotide difference with the SNP genotype in the middle of the 250-bp region. One construct contained the major allele of rs59532114 (Figure 5c), whereas the other contained the minor allele (Figure 5a). Compared with the major allele, the minor allele produced a 5-fold increase in luciferase expression (P < 0.001) in the human primary outer root sheath cells, suggesting that the rs59532114 minor allele results in increased transcriptional activity of the BCL2 promoter in these cells. In contrast, transfection into 293T and HEK001 cells resulted in a marginal, nonsignificant increase in transcriptional activity by t test, indicating that the increase in transcriptional activity may be cell-type specific (Figure 5c).
      Figure thumbnail gr5
      Figure 5SNP rs59532114 allele affects BCL2 transcriptional activity in primary human outer root sheath cells. (a) A 2.84-kb DNA region of the BCL2 promoter was cloned in dual-luciferase constructs. (b) rs59532114 major or minor allele residing in the middle of the 250-bp DNA sequence were tested. Firefly luciferase values were normalized to the cotransfected Renilla luciferase to correct transfection efficiency. (c) As compared with the wild-type or major allele (C), the minor allele (A) gave a significant increase in BCL2 transcriptional activity in human primary outer root sheath, but not in 293T and HEK001 cells. The error bar represents standard deviation among the replicates. (d) TNF-α up-regulates BCL2 expression in human primary outer root sheath cells. Microarray analysis was conducted after transient transfection. SNP, single-nucleotide polymorphism; TNF, tumor necrosis factor.

      TNF-α inhibition affects BCL2 expression through TNF signaling pathway

      We next examined possible mechanisms by which the adalimumab target, TNF, might influence BCL2 expression in HS disease–relevant tissues. Single-target knockdown of both TNF and BCL2, followed by genome-wide expression microarray analysis were performed. The same experiments were performed in both human primary outer root sheath cells and human primary keratinocytes. In the primary outer root sheath cells, we first confirmed that knockdown of TNF-α resulted in significantly decreased BCL2 expression (P = 1.71E–04) at the mRNA level (Figure 5d, left panel). In contrast, knockdown of BCL2 did not have a strong effect on TNF-α expression (P = 0.13) (Figure 5d, right panel), which suggests that adalimumab may down-regulate BCL2 expression partially through its effect on TNF-α, and suggests that BCL2 is downstream of TNF in a regulatory pathway in human primary outer root sheath cells.

      Discussion

      We have completed the largest pharmacogenomic GWAS of participants (N = 445 consented) with HS, investigating genetic variants associated with variation in response to adalimumab. We identified a single locus in BCL2, which suggested a potential role of apoptosis homeostasis in the pathophysiology of adalimumab response, in the context of HS disease. As the mechanistic link between associated SNPs and the response phenotype can often be obscure and requires experimental validation, we performed several functional genomics experiments to identify a mechanism implied by our GWAS association.
      The statistically most strongly associated variant, (rs5932114) was shown to be associated with increased expression and protein levels of BCL2 in LCLs. In addition, we demonstrated, using reporter assays, that the minor allele is also associated with increased BCL2 transcription in primary human hair outer root sheath cells, but not in other cell types. The knockdown of TNF and BCL2 separately showed that BCL2 belongs to the same pathway and is down-regulated by TNF. BCL2 acts as an antiapoptotic regulatory protein that blocks cell death and plays an essential role in regulating skin homeostasis in the outer root sheath cells of the hair follicles (
      • Abe Y.
      • Tanaka N.
      Roles of the hedgehog signaling pathway in epidermal and hair follicle development, homeostasis, and cancer.
      ). It has been reported in patients with psoriasis, based on lesional skin immunohistochemical staining, that another TNF-α inhibitor (infliximab) also regulated BCL2 protein through TNF and that TNF was upstream of BCL2 (
      • Kokolakis G.
      • Giannikaki E.
      • Stathopoulos E.
      • Avramidis G.
      • Tosca A.D.
      • Krüger-Krasagakis S.
      Infliximab restores the balance between pro- and anti-apoptotic proteins in regressing psoriatic lesions.
      ). Moreover, BCL2 has recently been shown to be associated with skin-related excessive hairiness (hirsutism) among Japanese (
      • Endo C.
      • Johnson T.A.
      • Morino R.
      • Nakazono K.
      • Kamitsuji S.
      • Akita M.
      • et al.
      Genome-wide association study in Japanese females identifies fifteen novel skin-related trait associations.
      ). Genetic mapping identified BCL2 as one of the regions controlling eccrine gland and hair follicle traits response for sweat gland density in mice (
      • Kamberov Y.G.
      • Karlsson E.K.
      • Kamberova G.L.
      • Lieberman D.E.
      • Sabeti P.C.
      • Morgan B.A.
      • et al.
      A genetic basis of variation in eccrine sweat gland and hair follicle density.
      ).
      As in many other GWAS with pharmacogenomics drug response, our study has several limitations (
      • Park H.W.
      • Dahlin A.
      • Tse S.
      • Duan Q.L.
      • Schuemann B.
      • Martinez F.D.
      • et al.
      Genetic predictors associated with improvement of asthma symptoms in response to inhaled corticosteroids.
      ). Our sample size is modest; however, our samples were selected from the largest two HS trials that have been performed (Figure 3). Critically, we deployed functional experiments to evaluate molecular mechanisms that could account for how these genetic variations affect the BCL2 gene. Our results provide multiple lines of supportive evidence for a plausible mechanism to support this statistical association. Together with these findings, our results support the hypothesis that apoptotic balance may be an important component of treatment response in HS and possibly other immune-mediated skin diseases. More specifically, this study provides evidence of a genetic link to adalimumab response and indicates that the key antiapoptotic factor, BCL2, may be repressed in HS-relevant tissues by anti-TNF agents (Supplementary Figure S3). These results broaden our understanding of underlying disease mechanisms in HS and may lead to the pursuit of additional mechanisms relevant to the treatment of HS either alone or in combination with anti-TNF agents.

      Materials and Methods

      Study design

      This study is based on two placebo-controlled randomized clinical trials, the Pioneer I (M11-313) and Pioneer II (M11-810) phase 3 clinical trials (NCT01468207 and NCT01468233, respectively;
      • Kimball A.B.
      • Okun M.M.
      • Williams D.A.
      • Gottlieb A.B.
      • Papp K.A.
      • Zouboulis C.C.
      • et al.
      Two phase 3 trials of adalimumab for hidradenitis suppurativa.
      ). Following written informed consent and Ethics Committee approvals, 445 trial participants consented DNA samples (199 from PIONEER I and 246 from PIONEER II) and were used for genotyping.
      In both trials, the primary efficacy end point was the percentage of participants achieving Hidradenitis Suppurative Clinical Response (HiSCR) at week 12. HiSCR is a binary outcome that is achieved when there is at least a 50% reduction in abscess and inflammatory nodule count (AN.COUNT) with no increase in abscess count and no increase in draining fistula compared with baseline (
      • Kimball A.B.
      • Sobell J.M.
      • Zouboulis C.C.
      • Gu Y.
      • Williams D.A.
      • Sundaram M.
      • et al.
      HiSCR (hidradenitis suppurativa clinical response): a novel clinical endpoint to evaluate therapeutic outcomes in patients with hidradenitis suppurativa from the placebo-controlled portion of a phase 2 adalimumab study.
      ). PIONEER I, achieved a significant difference in response rates at week 12, with 41.8% of participants on adalimumab achieving HiSCR compared with 29.8% of placebo. Similarly, in PIONEER II, a significant difference in response rates was observed (58.9% for treated vs. 36.8% for placebo.)

      Combining participants from four separate study arms

      Both PIONEER I and II consisted of three periods where some participants who initially received placebo were switched to adalimumab at later periods (Figure 1a). In period 1 (24 weeks) of both trials, participants were randomized in a 1:1 ratio to receive either 40 mg adalimumab every week or placebo. In period 2 of PIONEER I, participants who had been on placebo during period 1 were switched to adalimumab 40 mg EW. In PIONEER II, participants on placebo in period 1 continued on placebo until week 36, at which time (period 3) they were switched to adalimumab 40 mg every week in an open-label extension study. This study design resulted in four different groups that effectively received an initial 12-week adalimumab 40-mg every week dosing and efficacy determination. We combined these four treatment arms into a single GWAS analysis to maximize statistical power.

      Response definition

      HiSCR was used as a measure of response in the clinical trials, which as a classifier has reduced the statistical power of the GWAS analysis. The main quantitative component of HiSCR, AN.COUNTFC was defined and calculated as AN.COUNTWeek12 / AN.COUNTBaseline, where baseline was the first week of dosing. The data were normalized using a Log2 transformation, and this transformed metric was truncated at 2 to reduce the impact of several extreme outliers (with Log2 FC > 2). However, we found that untransformed FCs did not meet the assumptions of normal residuals in our statistical tests.
      Linear regression analysis was used to test a null hypothesis of no association between AN.COUNTFC and variant with MAF ≥ 5% in our sample (implemented in PLINK 2.0 linear regression feature). Dosage-based genotypes were used as opposed to allelic and took values ranging from 0 to 2 (PLINK dosage feature). Observed minimum serum adalimumab concentration (Ctrough) was used as a covariate. Only individuals who had nonmissing phenotype, covariate values, and who received adalimumab treatment in one of the periods were used in the analysis.

      GWAS genotyping, quality control, and imputation

      Genotyping was performed using Illumina HumanOmniExpressExome v1.2b BeadChip (Illumina, San Diego, CA) containing 964,193 SNPs. Initial genotype calling was performed and analyzed on all batches and samples using Illumina‘s Genome Studio software and was exported as PLINK format (
      • Oros K.K.
      • Arcand S.L.
      • Bayani J.
      • Squire J.A.
      • Mes-Masson A.M.
      • Tonin P.N.
      • et al.
      Analysis of genomic abnormalities in tumors: a review of available methods for Illumina two-color SNP genotyping and evaluation of performance.
      ) for bioinformatic analysis.
      PyGenClean (
      • Lemieux Perreault L.P.
      • Provost S.
      • Legault M.A.
      • Barhdadi A.
      • Dubé M.P.
      pyGenClean: efficient tool for genetic data clean up before association testing.
      ) was used for performing initial quality control. At the sample quality control level, of the original 445 individuals samples, 57 individuals were excluded as genetic ancestry outliers.
      At the SNP (marker) quality-control level, of 964,193 initial directly genotyped SNPs, 53,161 SNPs were removed because of quality-control issues such as high missing genotype rate or deviations from Hardy-Weinberg Equilibrium (P < 1.0E–4).
      With the quality-control-filtered genotypes, Beagle v. 4.1 (
      • Browning B.L.
      • Browning S.R.
      Genotype imputation with millions of reference samples.
      ) was used to input genotypes for 31.6 million variants, or all variants called in phase 3 of 1000 Genomes project. These were further filtered for MAF > 0.05 to 6,949,858 variants for testing. Beagle was run on each chromosome separately to impute both missing genotypes at variants on the SNP chip and variants not typed on the chip. Finally, estimated minor allele dosages output from Beagle were converted to PLINK dosage format for GWAS analysis.

      Validation in different sample sources

      Because no current replication cohort exists to retest significant findings, we turned to several different data sources for confirmatory evidence of our association results. First, we compared AN.COUNTFC at two other time points (week 4 and week 8 measurements) in the same participants who were used in the initial GWAS analysis. Second, we used data from a subset of participants who, after 12 weeks of initial treatment, went through a 24 week washout period, followed by a return to adalimumab treatment and measurements of AN.COUNT at 4, 8, 12, 18, 24, 36, and 48 weeks after return to adalimumab treatment (i.e., so-called switchover placebo participants). In this second treatment period, we measured the decay of AN.COUNTFC and tested for significant GWAS associations.

      Expression quantitative trait loci (eQTL) analysis

      eQTL analysis focused on top variant rs59532114 and genes within ±500 kb (each 1-Mb wide). LCLs of defined genotype at rs59532114 were bought from Coriell Institute NIGMS Human Genetic Cell Repository (Coriell Institute, Camden, NJ), then plated on nonadherent 12-well plates at a density of 1 × 105 cells per well, without antibiotics. RNA was extracted after 48 hours of culture using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Quantitative PCR was performed using TaqMan Advanced Master Mix with predesigned Taqman probes (Thermo Fisher Scientific) for genes, including BCL2, PHLPP1, KDSR, VPS4B, and SERPINB5, and compared against the control gene INST4 (cycle mean = 22.97, standard deviation = 1.01). Relative quantification over the control gene was calculated for the value of 2-ΔΔCT to get the expression levels. The statistical significance between means was tested using a two-tailed Student t test, with equal variances assumed. Each experiment was conducted in triplicates. A P-value of <0.05 was considered significant.

      Western blot analysis

      Four LCLs of each homozygous genotype at rs59532114 were grown in 25 ml of RPMI with 10% fetal bovine serum for 72 hours. Protein was extracted, and concentration was measured by BCA assay (Thermo Fisher Scientific, Waltham, MA), followed by transfer to polyvinylidene fluoride according to the manufacturer's instructions (LI-COR, Lincoln, NE). Primary antibodies against BCL2 (Novus Biologicals, Littleton, CO), VPS4B (Novus Biologicals), PHLPP1 (Abcam, Cambridge, UK), KDSR (Abcam), SERPINB5 (Abcam), and ACTB (Abcam) were tested because they are all located within 1 Mb of the response associated linkage disequilibrium block. The protein bands were detected using the Odyssey infrared imaging system (LI-COR). IRDye 800CW and IRDye 680 (LI-COR) served as secondary antibodies. All western blot band quantification procedures were performed using the Image Studio Lite 3.1 software (LI-COR).

      Plasmid construction and reporter assays

      Promoter-driven luciferase reporter constructs were generated by insertion of gBlocks (Integrated DNA Technologies, Coralville, IA), containing the BCL2 2.84 Kb promoter, into the KpnI/MluI sites of pGL3-Basic (Promega, Madison, WI). For the SNP rs59532114 locus, a 250-bp DNA with the SNP major allele C and the minor allele A in the middle were synthesized and cloned into the BamHI/SalI sites of BCL2-promoter construct.
      Human primary outer root sheath cells, HEK-293T or human primary keratinocyte cells were transfected with 500 ng luciferase reporter plasmids and 50 ng of pRL-TK transfection control plasmid with Lipofectamine 2000 (Thermo Fisher Scientific). The total amount of transfected DNA was kept constant at 500 ng for each construct. Luciferase activity was measured 48 hours after transfection assayed with a dual-luciferase system and normalized to renilla activity (Promega).

      Conflict of Interest

      The studies were sponsored by AbbVie. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the publication. Several authors are AbbVie employees and may hold AbbVie stocks or options.
      ML, JD, RWG, AN, NMM, HDT, DAW, JWD, and JFW are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
      AJN and RSK have served as a consultant for AbbVie. They were not funded by AbbVie.

      Acknowledgments

      The authors thank the participants for their participation and desire to provide their DNA for genetic research in the M11–313 and M11–810 studies. The authors also wish to thank Jozef Lazar, MD, PhD, of AbbVie for reviewing the manuscript.

      Author Contributions

      Conceptualization: ML, JD, NMM, HDT, DAW, RSK, AJN, JFW; Data Curation: ML, JD, RWG, AN, NMM, HDT, DAW, RSK, AJN, JWD, JFW; Formal Analysis: JD, JWD; Funding Acquisition: ML, JFW; Investigation: ML, JD, RGW, AN, NMM, HDT, DAW, JFW.

      Supplementary Material

      Figure thumbnail fx1
      Supplementary Figure S1The plot of the association between rs59532114 genotype carriers with baseline patients’ Hurley stage. There was no significant association to baseline disease severity as measured by AN count (P = 0.69). We additionally checked for an association between genotype and Hurley stage but found that there was an equal split between Hurley stage II and Hurley stage III subjects in the two genotype classes (CC and CA) of rs59532114 (55% Hurley stage II in homozygote major allele genotypes vs. 57% Hurley Stage II in heterozygotes; P = 1).
      Figure thumbnail fx2
      Supplementary Figure S2The minor allele of rs59532114 genotype-carried LCLs associates with increased BCL2 mRNA expression in African American and Han Chinese American LCLs data in published GEO dataset (GSE24277). Affymetrix microarray U133 results of BCL2 expression based on different genotype classes of LCLs were extracted from 100 African American LCLs (left) and 100 Han Chinese American LCLs (right). The heterozygous genotype of minor allele (Het WV) genotype was associated with increased mRNA expression of BCL2 from published GEO data (GSE24277). GEO, Gene Expression Omnibus; LCL, lymphoblastoid cell line.
      Figure thumbnail fx3
      Supplementary Figure S3In patients with HS, with BCL-2 in the TNF pathways of pathogenesis (left panel). Under adalimumab treatment, the homozygous of major allele (CC) rs59532114 carriers associate with decreased BCL2 expression and decreased antiapoptosis responses to adalimumab (middle panel). However, the minor allele (CA or AA) of rs59532114 carriers associated with increased BCL-2 expression and with strong antiapoptosis with lack of response to adalimumab treatment (right panel). HS, hidradenitis suppurativa; TNF, tumor necrosis factor.

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