Rare Loss-of-Function Mutation in SERPINA3 in Generalized Pustular Psoriasis
2020; Elsevier BV; Volume: 140; Issue: 7 Linguagem: Inglês
10.1016/j.jid.2019.11.024
ISSN1523-1747
AutoresSilke Frey, Heinrich Sticht, Dagmar Wilsmann‐Theis, Anne Gerschütz, Katharina Wolf, Sabine Löhr, Stefan Haskamp, Benjamin Frey, Madelaine Hahn, Arif B. Ekici, Steffen Uebe, Christian T. Thiel, André Reis, Harald Burkhardt, Frank Behrens, Michaela Köhm, Jürgen Rech, Georg Schett, G. Aßmann, Külli Kingo, Sulev Kõks, Rotraut Mößner, Jörg C. Prinz, Vinzenz Oji, Peter Schulz, Luis E. Muñoz, Andreas E. Kremer, Joerg Wenzel, Ulrike Hüffmeier,
Tópico(s)Psoriasis: Treatment and Pathogenesis
ResumoGeneralized pustular psoriasis (GPP) represents a rare pustular disease manifesting as a multisystemic inflammation in a chronic or episodic course, sometimes as life-threatening incidents. Biallelic IL36RN mutations are known to be disease-causing or -contributing in 21–41% of patients with GPP (Hussain et al., 2015Hussain S. Berki D.M. Choon S.E. Burden A.D. Allen M.H. Arostegui J.I. et al.IL36RN mutations define a severe autoinflammatory phenotype of generalized pustular psoriasis.J Allergy Clin Immunol. 2015; 135: 1067-1070.e9Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Marrakchi et al., 2011Marrakchi S. Guigue P. Renshaw B.R. Puel A. Pei X.Y. Fraitag S. et al.Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.N Engl J Med. 2011; 365: 620-628Crossref PubMed Scopus (668) Google Scholar, Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar, Onoufriadis et al., 2011Onoufriadis A. Simpson M.A. Pink A.E. Di Meglio P. Smith C.H. Pullabhatla V. et al.Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis.Am J Hum Genet. 2011; 89: 432-437Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, Sugiura et al., 2013Sugiura K. Takemoto A. Yamaguchi M. Takahashi H. Shoda Y. Mitsuma T. et al.The majority of generalized pustular psoriasis without psoriasis vulgaris is caused by deficiency of interleukin-36 receptor antagonist.J Invest Dermatol. 2013; 133: 2514-2521Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). IL-36 binds to the IL-36 receptor complex, thereby triggering the activation of mitogen-activated protein kinase and NF-κB, resulting in the production of proinflammatory cytokines. The IL-36 receptor antagonist competitively binds to the IL-36 receptor, inhibiting signal transduction (Hahn et al., 2017Hahn M. Frey S. Hueber A.J. The novel interleukin-1 cytokine family members in inflammatory diseases.Curr Opin Rheumatol. 2017; 29: 208-213Crossref PubMed Scopus (39) Google Scholar). IL36RN mutations lead to reduced antagonism of the IL-36 receptor, with an imbalance in the IL-36 pathway supportive of proinflammatory IL-36α, -β, and -γ cytokines (Marrakchi et al., 2011Marrakchi S. Guigue P. Renshaw B.R. Puel A. Pei X.Y. Fraitag S. et al.Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.N Engl J Med. 2011; 365: 620-628Crossref PubMed Scopus (668) Google Scholar). Additional disease-associated variants in CARD14 and/or AP1S3 were identified in 15% of patients carrying IL36RN mutations, suggesting an oligogenic rather than monogenic inheritance (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar). Because variants in known susceptibility genes cannot be identified in 64% of patients with GPP (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar), we aimed to detect more disease-relevant variants by performing whole exome sequencing in 25 patients with GPP. Our studies were approved by the ethical committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg and the University of Göttingen. Written informed consent was obtained from each patient and healthy volunteer before enrollment. Whole exome sequencing did not reveal any new candidate gene with autosomal-recessive inheritance in ≥2 patients. As effects of truncating variants can be predicted more reliably than effects of nonsynonymous variants, we selected genes with truncating variants in ≥2 patients. By analyzing gene function and expression, we prioritized SERPINA3 as the most relevant gene. We identified the same heterozygous deletion c.966delT/p.Tyr322Ter in two patients with GPP and confirmed it by Sanger sequencing (Supplementary Figure S1a and b). This rare variant was significantly associated with GPP (Pc = 2.65E-04, Table 1). Haplotype analyses indicated that the mutation localizes to the same common haplotype, leaving open whether the variant arose once or recurrently. Sanger sequencing of a further 54 patients with GPP, acrodermatitis continua suppurativa Hallopeau, or acute generalized exanthematous pustulosis revealed a rare heterozygous missense variant c.1046C>A/p.Ala349Asp (Supplementary Table S1).Table 1Number of Absolute Alleles and Frequencies of Rare Indel Variants in SERPINA3 in Patients with Pustular Psoriasis and Control Individuals and Results of Comparisons of Allele Frequency DistributionsVariant at nucleotide/ protein levelPosition in hg19 (dbSNP-ID)79 patients with GPP/ACH/AGEP398 patients with GPP/ACH/AGEP/PPP64,583 Non-Finnish European individuals (gnomAD)mutant – no. of alleles (%)wildtype – no. of alleles (%)mutant – no. of alleles (%)wildtype – no. of alleles (%)mutant – no. of alleles (%)wildtype – no. of alleles (%)c.966delT/p.Tyr322Terchr14:95,088,726 (rs771543687)2 (1.27)156 (98.73)2 (0.25)794 (99.75)8 (0.01)129,166 (99.99)Pc2.65E-040.0064OR (95% CI)207.52 (21.24–1011.41)40.65 (4.20–204.93)319 patients with PPP398 patients with GPP/ACH/AGEP/PPP64,523 Non-Finnish European individuals (gnomAD)c.221_223delTCT/p.Phe75delchr14:95,080,999-95,081,001 (rs748990999)1 (0.16)637 (99.84)1 (0.13)795 (99.87)14 (0.01)129,046 (99.99)Pc0.2840.352OR (95% CI)14.47 (0.34–95.39)11.59 (0.27–76.48)Abbreviations: ACH, acrodermatitis continua suppurativa Hallopeau; AGEP, acute generalized exanthematous pustulosis; GPP, generalized pustular psoriasis; OR, odds ratio; PPP, palmoplantar pustular psoriasis.Patients tested included 79 patients with GPP/ACH/AEP or 319 PPP patients or combined study groups. Control individuals were non-Finnish Europeans from gnomAD (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Google Scholar). Comparisons were made using Fisher's exact test. Open table in a new tab Abbreviations: ACH, acrodermatitis continua suppurativa Hallopeau; AGEP, acute generalized exanthematous pustulosis; GPP, generalized pustular psoriasis; OR, odds ratio; PPP, palmoplantar pustular psoriasis. Patients tested included 79 patients with GPP/ACH/AEP or 319 PPP patients or combined study groups. Control individuals were non-Finnish Europeans from gnomAD (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Google Scholar). Comparisons were made using Fisher's exact test. SERPINA3 encodes serine protease inhibitor A3 (serpin A3), which specifically inhibits several proteases (Cooperman et al., 1993Cooperman B.S. Stavridi E. Nickbarg E. Rescorla E. Schechter N.M. Rubin H. Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex.J Biol Chem. 1993; 268: 23616-23625Abstract Full Text PDF PubMed Google Scholar, Djie et al., 1997Djie M.Z. Stone S.R. Le Bonniec B.F. Intrinsic specificity of the reactive site loop of alpha 1-antitrypsin, alpha 1-antichymotrypsin, antithrombin III, and protease nexin I.J Biol Chem. 1997; 272: 16268-16273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The key functional site of serpin A3 is the reactive center loop, mediating binding of the target protease. After cleavage of the reactive center loop by the protease, a covalent serpin-protease complex is formed, leading to irreversible protease inactivation. Theoretically, the entire reactive center loop is missing in c.966delT/p.Tyr322Ter (Figure 1a), indicating a functionally inactive protein, whereas our in vitro experiments indicate nonsense-mediated mRNA decay and therefore haploinsufficiency of SERPINA3. The effect of c.1046C>A/p.Ala349Asp was predicted to be uncritical because of the residue's orientation to the solvent. Our quantitative expression analyses revealed the most abundant expression of SERPINA3 in the liver and lower expression in skin, dermal fibroblasts, HaCaT cells, trachea, and lung (Supplementary Figure S2a). In controls, seral serpin A3 was in the range of 0.05–0.23 mg/ml, the level in the two frameshift variant carriers at the range's low end (Figure 1b), matching the finding of a haploinsufficient gene. In male controls, we observed evidence for some correlation of serpin A3 with age (Figure 1c), confirming previous results (Herman et al., 2009Herman W.A. Seńko A. Korczowska I. Lacka K. Assessment of selected serum inflammatory markers of acute phase response and their correlations with adrenal androgens and metabolic syndrome in a population of men over the age of 40.Pol Arch Med Wewn. 2009; 119: 704-711PubMed Google Scholar). More importantly, the finding underlines the relevance of very low levels of serpin A3 in our 75-year-old male patient with GPP. We observed highly reduced mRNA expression after transfection of the mutant vector compared with the wildtype (Figure 1d), indicating nonsense-mediated mRNA decay. In the supernatants of cells transfected with the mutant, we could not detect any protein (Figure 1e). Immunohistochemistry revealed a confined expression of serpin A3 in the upper epidermis in patients with GPP carrying the frameshift variant, more pronounced at the edge of pustules (Figure 1f–h). As serpin A3 belongs to the acute phase proteins (Aronsen et al., 1972Aronsen K.F. Ekelund G. Kindmark C.O. Laurell C.B. Sequential changes of plasma proteins after surgical trauma.Scand J Clin Lab Invest Suppl. 1972; 124: 127-136Crossref PubMed Scopus (265) Google Scholar), we stimulated SERPINA3's expression in several cell lines. In HaCaT cells, we observed an earlier stimulation, whereas induction was highest in dermal fibroblasts (Supplementary Figure S2b–e). Sequencing analysis of SERPINA3 by Sanger in 319 patients with palmoplantar pustular psoriasis revealed two rare heterozygous coding variants. The effect of c.8G>A/p.Arg3Lys was predicted to be negligible, whereas the other variant's (in-frame-deletion c.221_223delTCT/p.Phe75del) effect was a significant conformational rearrangement of the adjacent residues of the shutter region (Supplementary Figure S3, Supplementary Results). c.221_223delTCT/p.Phe75del was not significantly associated with palmoplantar pustular psoriasis (Table 1). A targeted analysis of the frameshift variant in 2,223 patients with psoriasis vulgaris and psoriatic arthritis did not identify a single carrier. Serpin A3's interaction with cathepsin G is the strongest among the proteases (Beatty et al., 1980Beatty K. Bieth J. Travis J. Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin.J Biol Chem. 1980; 225: 3931-3934Abstract Full Text PDF Google Scholar). In vitro analyses indicated that neutrophilic serine proteases including cathepsin G can escalate inflammation by cleavage leading to an increased activation of less active IL-36β by a factor >500 (Henry et al., 2016Henry C.M. Sullivan G.P. Clancy D.M. Afonina I.S. Kulms D. Martin S.J. Neutrophil-derived proteases escalate inflammation through activation of IL-36 family cytokines.Cell Rep. 2016; 14: 708-722Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) (Supplementary Figure S4a). In an acute GPP episode with infiltration of neutrophil granulocytes in the upper epidermis, there is a close proximity of neutrophils to serpin A3. The known inhibitory effect of serpin A3 on cathepsin G suggests a regulatory interaction of both proteins in normal skin and an imbalance in the case of patients with GPP carrying SERPINA3 mutations (Supplementary Figure S4b). SERPINA3 has been identified to be expressed at higher levels in psoriasis vulgaris skin than normal skin (Li et al., 2014Li B. Tsoi L.C. Swindell W.R. Gudjonsson J.E. Tejasvi T. Johnston A. et al.Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms.J Invest Dermatol. 2014; 134: 1828-1838Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Based on our own experiences (D'Erme et al., 2015D'Erme A.M. Wilsmann-Theis D. Wagenpfeil J. Hölzel M. Ferring-Schmitt S. Sternberg S. et al.IL-36gamma (IL-1F9) is a biomarker for psoriasis skin lesions.J Invest Dermatol. 2015; 135: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), this differentiates psoriasis vulgaris from further chronic skin diseases such as atopic dermatitis and lichen planus without upregulated SERPINA3. A more recent comparison of the transcriptome in GPP skin with psoriasis vulgaris skin indicated that serpin A3 was upregulated in both psoriatic subtypes (Johnston et al., 2017Johnston A. Xing X. Wolterink L. Barnes D.H. Yin Z. Reingold L. et al.IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis.J Allergy Clin Immunol. 2017; 140: 109-120Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Although serum amount was in the lowest normal range in both carriers of c.966delT/p.Tyr322Ter, serpin A3 was determined in affected, but not normal, epidermis. This disparity might be explained by different disease phases, an acute GPP episode with upregulation of acute phase proteins during skin biopsy in contrast to the healthy interval of blood sampling. Lack of differences in serum levels of IL-36α and IL-36β between patients and controls (Supplementary Figure S5) might also reflect the healthy interval or lack of sufficiently sensitive ELISAs. In conclusion, we detected a loss-of-function variant in SERPINA3 in two independent patients, and in combination with our experimental data, we propose SERPINA3 as a new candidate gene for GPP. The small fraction of patients carrying mutations in SERPINA3 fits the known heterogeneity in GPP, and the inherited variant by a healthy parent in one case would be in accordance with the previously suggested oligogenic inheritance pattern. Genetic studies of independent patient groups are necessary to replicate our findings, and further experimental studies will shed light on SERPINA3's role in GPP and other pustular psoriasis subtypes. Whole exome sequencing was performed to detect new candidate genes in GPP. Because of privacy regulations, the whole datasets are not publicly available, but authors can be contacted for specific issues. Silke Frey: http://orcid.org/0000-0001-9411-8357 Heinrich Sticht: http://orcid.org/0000-0001-5644-045X Dagmar Wilsmann-Theis: http://orcid.org/0000-0002-5594-7192 Anne Gerschütz: http://orcid.org/0000-0002-3202-562X Katharina Wolf: http://orcid.org/0000-0001-7931-6056 Sabine Löhr: http://orcid.org/0000-0001-7720-0762 Stefan Haskamp: http://orcid.org/0000-0003-3760-9074 Benjamin Frey: http://orcid.org/0000-0001-6743-3351 Madelaine Hahn: http://orcid.org/0000-0001-7028-0456 Arif B. Ekici: http://orcid.org/0000-0001-6099-7066 Steffen Uebe: http://orcid.org/0000-0002-4819-7637 Christian Thiel: http://orcid.org/0000-0003-3817-7277 André Reis: http://orcid.org/0000-0002-6301-6363 Harald Burkhardt: http://orcid.org/0000-0002-6261-3131 Frank Behrens: http://orcid.org/0000-0001-8750-7186 Michaela Köhm: http://orcid.org/0000-0002-3579-5574 Jürgen Rech: http://orcid.org/0000-0002-2569-2029 Georg Schett: http://orcid.org/0000-0001-8740-9615 Gunter Assmann: http://orcid.org/0000-0002-3216-9340 Külli Kingo: http://orcid.org/0000-0001-6301-9612 Sulev Kõks: http://orcid.org/0000-0001-6087-6643 Rotraut Mössner: http://orcid.org/0000-0003-2476-7647 Jörg C. Prinz: http://orcid.org/0000-0002-8857-5495 Vinzenz Oji: http://orcid.org/0000-0003-1380-4828 Peter Schulz: http://orcid.org/0000-0002-3168-2660 Luis E. Muñoz: http://orcid.org/0000-0002-5395-804X Andreas E. Kremer: http://orcid.org/0000-0002-9263-948X Jörg Wenzel: http://orcid.org/0000-0002-4744-5993 Ulrike Hüffmeier: http://orcid.org/0000-0001-6448-4671 KK is an investigator for Celgene, Merck, Mitsubishi Pharma, Novartis, Regeneron Pharmaceuticals, Inc., and Sandoz. All other authors state no conflict of interest. The authors are grateful to all patients and healthy volunteers for participation in this project. The authors thank Ingo Ganzleben and Christoph Becker for providing the cell-line BEAS-2B (epithelial of lung bronchus). This project was partly funded by a grant to SF and UH from the DFG (CRC1181, project A05), by a grant to AR and UH from the BMBF (Metarthros 01EC1407A), by grants to UH from the DFG (HU 2163/1-1) and from the Interdisciplinary Centre for Clinical Research (laboratory rotation) of the Clinical Center Erlangen of the Friedrich-Alexander-Universität Erlangen-Nürnberg , and by support of the Innovative Medicine Initiative (IMI RTCure). Formal Analysis: HS, AG, KW, SL, SH, MH, SU; Funding Acquisition: UH; Investigation: SF, AG, KW, SL, MH; Methodology: AG, SH, BF, LEM; Project Administration: AG, UH; Resources: SF, DWT, HB, FB, MK, JR, GS, GA, KK, SK, RM, JP, AK, VO, PS, AEK; Software: HS, ABE, CT, SU; Supervision: SF, UH, AEK, JW; Validation: AG, SL; Visualization: SH, JW, UH; Writing - Original Draft Preparation: UH; Writing - Review and Editing: SF, AR, AEK We sequenced whole exomes of 25 patients with generalized pustular psoriasis (GPP), published within a larger group previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar). Those patients were selected based on a negative carrier status of IL36RN mutations. In addition, we used DNAs of 47 patients with independent GPP, 3 patients with acute generalized exanthematous pustulosis, and 4 patients with acrodermatitis continua suppurativa Hallopeau—also described previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar)—for a targeted SERPINA3 analysis. We also investigated DNAs of 273 German and 46 Estonian patients with palmoplantar pustular psoriasis (PPP) and 1,099 patients with psoriasis vulgaris, 1,124 patients with psoriatic arthritis, and 934 control individuals, described in similar compositions previously (Löhr et al., 2019Löhr S. Ekici A.B. Uebe S. Büttner C. Köhm M. Behrens F. et al.Association analyses of psoriatic arthritis and psoriasis vulgaris with functional NCF1 variants.Rheumatology(Oxford). 2019; 58: 915-917Crossref PubMed Scopus (3) Google Scholar, Mössner et al., 2015Mössner R. Frambach Y. Wilsmann-Theis D. Löhr S. Jacobi A. Weyergraf A. et al.Palmoplantar pustular psoriasis is associated with missense variants in CARD14, but not with loss-of-function mutations in IL36RN in European patients.J Invest Dermatol. 2015; 135: 2538-2541Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The investigations were conducted according to Declaration of Helsinki principles. All DNAs used for whole exome sequencing passed quality control and were enriched using the SureSelect Human All Exons Kits (version 4 [n = 12] or version 5 [n = 13], Agilent, Santa Clara, CA). Sequencing was performed on a SOLiD 5500 XL (n = 19) (Thermo Fisher Scientific, Waltham, MA) or an Illumina HiSeq 2500 (n = 6) (Illumina, San Diego, CA). Reads were aligned with the bwa mem algorithm (Li and Durbin, 2009Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (24737) Google Scholar) using the human genome assembly hg19 as a reference. Single nucleotide variants and small insertions and deletions were called using the algorithms GATK Haplotype Caller, GATK Universal Genotyper, SNVer, ATLAS2, diBayes, freeBayes, LifeScope InDel Caller, and Platypus and annotated with ANNOVAR (Wang et al., 2010Wang K. Li M. Hakonarson H.H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7264) Google Scholar). On average, coverage of the target was 144.1×, and 89.0% of the target sequence was covered ≥20×. We included variants with an allele frequency of ≤0.3% in all European individuals of the Exome Aggregation Consortium (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6277) Google Scholar) and an allele frequency of ≤0.5% in the Exome Variant Server and 1000 Genomes. Selected variants had coverage of ≥20× and were identified ≤2× in a group of 421 independent individuals sequenced in-house for projects analyzing nonimmunological diseases. All sequenced patients with GPP were independent individuals. We prioritized for variants in overlapping genes using different modes of inheritance. As mutations in IL36RN, the first major gene in GPP, have been identified on both alleles, we selected for homozygous or compound heterozygous variants in overlapping genes but were unable to identify a single gene. Therefore, we prioritized for heterozygous truncating variants and identified two truncating variants in six different genes (Supplementary Tables S2 and S3), which were further evaluated using the Integrative Genomics Viewer (Thorvaldsdóttir et al., 2013Thorvaldsdóttir H. Robinson J.T. Mesirov J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.Brief Bioinform. 2013; 14: 178-192Crossref PubMed Scopus (4562) Google Scholar). Function and expression of candidate genes were considered to further select one candidate gene. Variants in NLRP5 and SERPINA3 were confirmed by Sanger sequencing. Parents of one of the two patients were available for targeted testing of the SERPINA3 variant. Supplementary Table S4 gives an overview on rare variants in other genes in the two carriers of the SERPINA3 frameshift variant. Structural analysis of serine protease inhibitor A3 (serpin A3) was performed on the basis of the known crystal structure (PDB:1QMN) (Gooptu et al., 2000Gooptu B. Hazes B. Chang W.S. Dafforn T.R. Carrell R.W. Read R.J. et al.Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease.Proc Natl Acad Sci USA. 2000; 97: 67-72Crossref PubMed Scopus (175) Google Scholar). Variant p.Pro55Leu (p.Pro78Leu according to NM_001085) present in the structure was reverted to the wildtype using Swiss-PDBViewer (Guex and Peitsch, 1997Guex N. Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). ModLoop (Fiser and Sali, 2003Fiser A. Sali A. ModLoop: automated modeling of loops in protein structures.Bioinformatics. 2003; 19: 2500-2501Crossref PubMed Scopus (566) Google Scholar) was applied to model the deletion of phenylalanine and to add missing residues of the reactive center loop (PDB:7API) (Engh et al., 1989Engh R. Löbermann H. Schneider M. Wiegand G. Huber R. Laurell C.B. The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism.Protein Eng. 1989; 2: 407-415Crossref PubMed Scopus (55) Google Scholar). RasMol (Sayle and Milner-White, 1995Sayle R.A. Milner-White E.J. RASMOL: biomolecular graphics for all.Trends Biochem Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2298) Google Scholar) was used for visualization. We sequenced exons of SERPINA3 in 54 independent patients with rare pustular psoriasis (GPP, acrodermatitis continua suppurativa Hallopeau, and acute generalized exanthematous pustulosis) by Sanger (primer sequences, Supplementary Table S5; reference sequence, NM_001085). Furthermore, we established a multiplex ligation-dependent probe amplification for exons if possible or regions closed to exons (sequences of oligos, Supplementary Table S6) and performed multiplex ligation-dependent probe amplification in all patients with rare pustular psoriasis forms (n = 79). We applied Fisher's exact test to determine allele frequency differences between patients and control individuals as described previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar) and to obtain evidence for the association of combination of GPP, acrodermatitis continua suppurativa Hallopeau, and acute generalized exanthematous pustulosis and of pustular psoriasis in general with c.966delT/p.Tyr322Ter and for the association of PPP and of pustular psoriasis in general with c.221_223delTCT/p.Phe75del, correcting the P-value for Bonferroni (n = 4). For differential expression analyses, we used a multitissue panel (Clontech, Mountain View, CA) including RNAs collected from the following human tissues: bone marrow, whole brain, fetal brain, fetal liver, heart, kidney, liver, lung, placenta, prostate, skeletal muscle, spleen, testis, thymus, trachea, uterus, colon, small intestine, spinal cord, and stomach. In addition, we used human mRNA from a lymphoblastoid cell line, two skin biopsies described previously (Hüffmeier et al., 2009Hüffmeier U. Lascorz J. Becker T. Schürmeier-Horst F. Magener A. Ekici A.B. et al.Characterisation of psoriasis susceptibility locus 6 (PSORS6) in patients with early onset psoriasis and evidence for interaction with PSORS1.J Med Genet. 2009; 46: 736-744Crossref PubMed Scopus (24) Google Scholar), a HaCaT cell line (Cell Lines Service, Eppelheim, Germany), two liver cell lines (American Tissue Culture Collection, Manassas, Virginia), CD14+ cells (negative selection with magnetic beads [Stemcell Technologies, Cologne, Germany]), and CD16+ cells (cell separation with a ficoll gradient). Tissues were provided by healthy donors. We performed quantitative PCR of cDNAs for expression of SERPINA3 in different tissues using a predesigned assay (Hs01038298_m1; Thermo Fisher Scientific) including an exon-spanning probe as described previously (Löhr et al., 2019Löhr S. Ekici A.B. Uebe S. Büttner C. Köhm M. Behrens F. et al.Association analyses of psoriatic arthritis and psoriasis vulgaris with functional NCF1 variants.Rheumatology(Oxford). 2019; 58: 915-917Crossref PubMed Scopus (3) Google Scholar, Uebe et al., 2017Uebe S. Ehrlicher M. Ekici A.B. Behrens F. Böhm B. Homuth G. et al.Genome-wide association and targeted analysis of copy number variants with psoriatic arthritis in German patients.BMC Med Genet. 2017; 18: 92Crossref PubMed Scopus (4) Google Scholar). ACTB, B2M, and PKG1 were used as housekeeping genes. The amount of serpin A3 in sera was determined with a commercially available ELISA (KA2128, Abnova, Taipei City, Taiwan). After a test run using three different dilutions of control sera (1:1,000, 1:2,500, and 1:5,000), we selected a dilution of 1:5,000 for measurements in two patients and 13 healthy volunteers (5 males and 8 females). We performed immunohistochemistry of serpin A3 in the skin of patients with GPP carrying the SERPINA3 frameshift mutation and of controls by using antibodies provided by Abcam (Cambridge, United Kingdom). Immunofluorescence of the skin of patients and control probands was performed as described previously (D'Erme et al., 2015D'Erme A.M. Wilsmann-Theis D. Wagenpfeil J. Hölzel M. Ferring-Schmitt S. Sternberg S. et al.IL-36gamma (IL-1F9) is a biomarker for psoriasis skin lesions.J Invest Dermatol. 2015; 135: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). To induce expression of SERPINA3, we used 1 μM dexamethasone (Sigma-Aldrich/Merck, St. Louis, MO); the cytokines tumor necrosis factor-α, IL-6, and IL-1β (ImmunoTools, Friesoythe, Germany); and lipopolysaccharide. We induced expression with dexamethasone only or combinations of dexamethasone with other stimuli in skin-derived cell lines and single stimuli in Hep3B and Hep2G cells. Cells were harvested at 6 hours and 24 hours after incubation with different concentrations of stimuli. Levels of saturation of SERPINA3's induction were shown (Supplementary Figure 2b–e). A plasmid containing the open reading frame of the SERPINA3 gene (Hölzel, Cologne, Germany) was used for transfection of HaCaTs; its sequence was confirmed by Sanger. To introduce the 1 base pair deletion, we performed mutagenesis using QuikChange Site-Directed Kit (Agilent) (primers, Supplementary Table S7). HaCaTs were transfected with lipofectamine 3000 using an empty vector, wildtype plasmid, or 1 base pair–deletion plasmid. Transfection of HaCaTs was performed in three independent experiments in duplicates or triplicates. Cell nuclei were labeled with DAPI (Sigma-Aldrich/Merck), whereas constructs were detectable by orange fluorescent protein. We determined transfection efficiency in HaCaTs using Image J (https://imagej.nih.gov/ij/index.html). Cells stained with DAPI and with or without the vector's orange fluorescent protein were counted in three different images containing >900 cells per image. We ob
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