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Palmoplantar Pustular Psoriasis is Associated with Missense Variants in CARD14, but not with loSs-of-Function Mutations in IL36RN in European Patients Rotraut Mo¨ssner, Yvonne Frambach, Dagmar WilsmannTheis, Sabine Lo¨hr, Arnd Jacobi, Ansgar Weyergraf, Michael Mu¨ller, Sandra Philipp, Regina Renner, Heiko Traupe, Harald Burkhardt, Ku¨lli Kingo, Sulev Ko˜ks, Steffen Uebe, Michael Sticherling, Heinrich Sticht, Vinzenz Oji, Ulrike Hu¨ffmeier

Cite this article as: Rotraut Mo¨ssner, Yvonne Frambach, Dagmar WilsmannTheis, Sabine Lo¨hr, Arnd Jacobi, Ansgar Weyergraf, Michael Mu¨ller, Sandra Philipp, Regina Renner, Heiko Traupe, Harald Burkhardt, Ku¨lli Kingo, Sulev Ko˜ks, Steffen Uebe, Michael Sticherling, Heinrich Sticht, Vinzenz Oji, Ulrike Hu¨ffmeier, Palmoplantar Pustular Psoriasis is Associated with Missense Variants in CARD14, but not with loSs-of-Function Mutations in IL36RN in European Patients, Journal of Investigative Dermatology accepted article preview 19 May 2015; doi: 10.1038/jid.2015.186. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Accepted article preview online 19 May 2015

© 2015 The Society for Investigative Dermatology

Palmoplantar pustular psoriasis is associated with missense variants in CARD14, but not with loss-of-function mutations in IL36RN in European patients

Rotraut Mössner 1, Yvonne Frambach 2,3, Dagmar Wilsmann-Theis 4, Sabine Löhr 5, Arnd Jacobi 6, Ansgar Weyergraf 7, Michael Müller 8, Sandra Philipp 9, Regina Renner 10, Heiko Traupe 11, Harald Burkhardt 12, Külli Kingo 13, Sulev Kõks 14, Steffen Uebe 5, Michael Sticherling 10, Heinrich Sticht 15, Vinzenz Oji 11, Ulrike Hüffmeier 5

1 Department of Dermatology, Georg-August-University Göttingen, Göttingen, Germany 2 Universität zu Lübeck, Department of Dermatology, Lübeck, Germany 3 Hanse-Klinik, Lübeck, Germany 4 Department of Dermatology and Allergy, University Bonn, Bonn, Germany 5 Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 6 Institute for Health Services Research in Dermatology and Nursing, University Medical Center Hamburg-Eppendorf, Hamburg and Department of Dermatology, University Marburg, Marburg, Germany 7 Department of Dermatology, Fachklinik Bad Bentheim, Bad Bentheim, Germany 8 Institute of Occcupational, Social and Environmental Medicine, Georg-August-University Göttingen, Göttingen, Germany 9 Department of Dermatology and Allergy, Universitätsmedizin Berlin, Berlin, Germany 10 Department of Erlangen, Germany

Dermatology,

Friedrich-Alexander-Universität

Erlangen-Nürnberg,

11 Department of Dermatology, University Münster, Münster, Germany 12 Division of Rheumatology and IME-Fraunhofer Project Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany 13 Dermatology Clinic, Clinics of Tartu University and Department of Dermatology, University of Tartu, Tartu, Estonia 14 Department of Pathophysiology, University of Tartu, Tartu, Estonia 15 Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Germany

Corresponding author: PD Dr. Ulrike Hüffmeier, Institute of Human Genetics, FriedrichAlexander-Universität Erlangen-Nürnberg, Schwabachanlage 10, 90154 Erlangen, Germany, email: [email protected], Phone: +49 9131 85 24192, Fax: +49 9131 85 23232

© 2015 The Society for Investigative Dermatology

Abbreviations: PPP = palmoplantar pustular psoriasis, GPP = generalized pustular psoriasis, PsV = psoriasis vulgaris, PsA = psoriatic arthritis

Mössner et al.: PPP and missense variants in CARD14

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To the editor Psoriasis is a complex genetic disease, meaning that a combination of genetic and environmental risk factors leads to disease manifestation. The most common cutaneous manifestation is psoriasis vulgaris (PsV) which is strongly associated with an HLA-C risk allele located at psoriasis susceptibility locus 1 (PSORS1). This locus has a greater impact on PsV, while there are many other genetic risk factors, but most of these contribute far less. Only in some families and single patients suffering from PsV as well as in patients with a severe pustular variant of psoriasis, named generalized pustular psoriasis (GPP), few other major genetic risk factors responsible for those disease manifestations were identified. These include missense variants in CARD14 and loss-of-function mutations in IL36RN described to be responsible for PsV and GPP or GPP, respectively (Jordan et al., 2012a; Jordan et al., 2012b; Marrakchi et al., 2011; Onoufriadis et al., 2011). Palmoplantar pustular psoriasis, also termed palmoplantar pustulosis (PPP), is characterized by sterile pustules on the palms and soles. It is more frequent in women and has stronger association with tobacco smoking than PsV or psoriatic arthritis (PsA) (O'Doherty and MacIntyre, 1985). While no randomized controlled studies with biologics licensed for PsV have been published, conventional therapy is similar to PsV, although there are differences in therapeutic outcome (De Mozzi et al., 2012; Marsland et al., 2006). Little is known about genetic risk factors of PPP; however, an association of PPP with missense mutations in IL36RN has been described recently (Setta-Kaffetzi et al., 2013). There is an ongoing discussion on whether PPP is a pustular variant of psoriasis or a distinct disease entity (Ammoury et al., 2008; Brunasso et al., 2013). In order to characterize PPP genetically, we recruited a cohort of 251 PPP patients and screened them for the major genetic risk factor for PsV at PSORS1 as well as for the two genes IL36RN and CARD14 as described previously (Huffmeier et al., 2009; Korber et al., 2013). Approval for the studies was obtained through the ethical committees of the responsible universities. Individuals gave their written informed consent. European Mössner et al.: PPP and missense variants in CARD14

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Americans from publicly available databases, from a previous publication (Jordan et al., 2012a) and control individuals from a German cohort (Huffmeier et al., 2009) served as control groups (for slight variations in control individuals, please see Supplements). The majority of the 251 PPP patients was female (78.1%) and 88.4% of recruited patients had a history of smoking, with 75% smoking at the time of their first psoriatic manifestation (Table 1, Supplements). The percentages of patients with concomitant PsV and a positive family history for psoriatic manifestations were 16.6% and 32.5%, respectively, and 24.6% of PPP patients reported to have been diagnosed with PsA. Four of the 251 PPP patients were identified to carry previously identified IL36RN mutations by Sanger-sequencing (Table 2): one carried c.227C>T/ p.Pro76Leu in a heterozygous form and three further patients were heterozygous carriers of c.338C>T/ p.Ser113Leu, summing up to a carrier rate of 2.1%. In addition, a further patient was heterozygous for c.17C>T/ p.Ala6Val, a missense variant in IL36RN. To the best of our knowledge, this variant has not been observed before. Molecular modeling revealed that valine at the 6th amino acid position causes some minor local structural changes, but due to a neighboring disulfide-bridge formed by Cys8, the global structure of the N-terminus can be stabilized. The effect at protein level is probably marginal and thus the variant was excluded from further association analyses. In comparison to different control groups (Table 2), we observed no increased frequency of mutant alleles in IL36RN and therefore no evidence for association. Previously, association of PPP with IL36RN mutations has been described with an effect size (odds ratio (OR)) of 12.3 [5.8-25.8] (Setta-Kaffetzi et al., 2013). Our study had 80% power to detect effects sizes lower than the lower limit of the 95% confidence interval in that study. With our cohort we had >99.999% power to detect an association of PPP with IL36RN mutant alleles based on allele frequency and effect size reported previously (Setta-Kaffetzi et al., 2013). Sequencing of the first three coding exons of CARD14 in 251 PPP patients revealed several interesting coding variants: Six heterozygous carriers of c.599G>A/ p.Ser200Asn, and two heterozygous carriers of c.526G>C/ p.Asp176His were identified (Table 2). In two different Mössner et al.: PPP and missense variants in CARD14

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groups of 1,049 controls genotyped in-house, we identified additionally c.536G>A/ p.Arg179His in heterozygous state in one control individual and 10 heterozygous carriers of c.599G>A/ p.Ser200Asn or c.526G>C/ p.Asp176His. These missense variants have been previously described to be involved in PsV and GPP (Jordan et al., 2012a; Korber et al., 2013; Sugiura et al., 2014). In addition, we identified the missense variant c.589G>A/ p.Glu197Lys exclusively in three German controls, previously neither reported nor functionally tested. Therefore, we excluded this variant from further analyses. With regard to the three functionally tested variants and in comparison to additional publicly available data at the exome variant server (http://evs.gs.washington.edu/EVS/), within the 1000 genomes project (Genomes Project et al., 2010) (http://www.1000genomes.org/) and a previous control cohort of 1,609 control individuals (Jordan et al., 2012a), we observed a highly significant association (p = 3.81E-04) and a corresponding OR of 5.13 [2.08-11.04]. Of note, the effect size is considerably higher than the effect sizes described in PsV (Jordan et al., 2012a). Of the eight PPP patients carrying the three missense variants in CARD14 (Table 3, last column), one patient (12.5%) had concomitant PsV and PsA, another patient had concomitant PsA only. Among the PPP patients, neither the frequencies of PsV and PsA) nor the frequency of patients who had ever smoked or had a history of smoking differed between carriers and non-carriers of the three CARD14 missense variants. Both the percentage of male patients carrying a CARD14 variant and the percentage of patients with negative family history for a psoriatic manifestation were higher compared to PPP patients without CARD14 missense variants. However, these results have to be interpreted with caution due to the small size of the groups. With regard to the PSORS1 risk allele, we observed no significant differences in carrier frequencies between PPP patients and healthy controls (Table 2). We had 80% power to detect an OR of at least 1.73 given the observed allele frequency. This effect size is considerably lower than the one observed previously in our large cohorts of PsV and PsA

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patients: OR = 8.1 [6.5–10.1] for PsV and OR = 4.5 [3.6–5.7] for PsA. Our negative association finding is in concordance to previous studies (Asumalahti et al., 2003; Fan et al., 2007; Karvonen et al., 1976). Overall, our investigations suggest an involvement of missense variants in CARD14 in the pathogenesis of PPP, but do not support a role for IL36RN mutations. These results suggest that genetic risk factors in PPP overlap with PsV and GPP, while we hypothesize that there will be additional, so far unknown, genetic risk factors probably restricted to PPP that are responsible for the typical, locally restricted palmoplantar pustular manifestation.

Conflict of Interest The authors declare that there is no conflict of interest.

Acknowledgements We acknowledge all patients and control probands for participation in this study making it possible. We thank Daniela Hemmeter and Anne Gerschütz for excellent technical assistance. Parts of this study were supported by two different grants to U.H.: a) from the German Research Foundation (DFG 2163/1-1) and b) from the ELAN funding of the Friedrich-Alexander-Universität

Erlangen-Nürnberg

(“Erlanger

Leistungsbezogene

Anschubfinanzierung und Nachwuchsförderung”, ELAN-13-03-09-1-UH). The contribution of H.B. was supported by the Federal State of Hesse (LOEWE-Project: Fraunhofer IME-ProjectGroup Translational Medicine and Pharmacology, Goethe University Frankfurt).

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References Ammoury A, El Sayed F, Dhaybi R, et al. (2008) Palmoplantar pustulosis should not be considered as a variant of psoriasis. Journal of the European Academy of Dermatology and Venereology : JEADV 22:392-3. Asumalahti K, Ameen M, Suomela S, et al. (2003) Genetic analysis of PSORS1 distinguishes guttate psoriasis and palmoplantar pustulosis. The Journal of investigative dermatology 120:627-32. Brunasso AM, Puntoni M, Aberer W, et al. (2013) Clinical and epidemiological comparison of patients affected by palmoplantar plaque psoriasis and palmoplantar pustulosis: a case series study. The British journal of dermatology 168:1243-51. De Mozzi P, Johnston GA, Alexandroff AB (2012) Psoriasis: an evidence-based update. Report of the 9th evidenced based update meeting, 12 May 2011, Loughborough, UK. The British journal of dermatology 166:252-60. Fan X, Yang S, Sun LD, et al. (2007) Comparison of clinical features of HLA-Cw*0602positive and -negative psoriasis patients in a Han Chinese population. Acta dermatovenereologica 87:335-40. Genomes Project C, Abecasis GR, Altshuler D, et al. (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061-73. Huffmeier U, Lascorz J, Bohm B, et al. (2009) Genetic variants of the IL-23R pathway: association with psoriatic arthritis and psoriasis vulgaris, but no specific risk factor for arthritis. The Journal of investigative dermatology 129:355-8. Jordan CT, Cao L, Roberson ED, et al. (2012a) Rare and common variants in CARD14, encoding an epidermal regulator of NF-kappaB, in psoriasis. American journal of human genetics 90:796-808. Jordan CT, Cao L, Roberson ED, et al. (2012b) PSORS2 is due to mutations in CARD14. American journal of human genetics 90:784-95. Karvonen J, Tilikainen A, Lassus A (1976) HLA antigens in psoriasis. A family study. Annals of clinical research 8:298-304. Korber A, Mossner R, Renner R, et al. (2013) Mutations in IL36RN in patients with generalized pustular psoriasis. The Journal of investigative dermatology 133:2634-7. Marrakchi S, Guigue P, Renshaw BR, et al. (2011) Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis. The New England journal of medicine 365:620-8. Marsland AM, Chalmers RJ, Hollis S, et al. (2006) Interventions for chronic palmoplantar pustulosis. Cochrane database of systematic reviews:CD001433. O'Doherty CJ, MacIntyre C (1985) Palmoplantar pustulosis and smoking. British medical journal 291:861-4. Onoufriadis A, Simpson MA, Pink AE, et al. (2011) Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. American journal of human genetics 89:432-7. Mössner et al.: PPP and missense variants in CARD14

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Setta-Kaffetzi N, Navarini AA, Patel VM, et al. (2013) Rare pathogenic variants in IL36RN underlie a spectrum of psoriasis-associated pustular phenotypes. The Journal of investigative dermatology 133:1366-9. Sugiura K, Muto M, Akiyama M (2014) CARD14 c.526G>C (p.Asp176His) is a significant risk factor for generalized pustular psoriasis with psoriasis vulgaris in the Japanese cohort. The Journal of investigative dermatology 134:1755-7.

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Table 1: Clinical characteristics of PPP patients Numbers indicate absolute counts (and percentages). PPP = PPP patients, Y = yes, N = no.

Category

Subcategory

251 PPP

217 German PPP

34 Estonian PPP

8 PPP carrying CARD14 variants

51.1±12.9

50.6±13.1

54.7±11.1

54.4±14.5

41.8±13.6

41.4±12.6

49.0±10.2

45.7±16.9

Average age at recruitment Average age at st 1 psoriatic manifestation Gender male/ female

male female

55 (21.9) 196 (78.1)

47 (21.7) 170 (78.3)

8 (23.5) 26 (76.5)

3 (37.5) 5 (62.5)

Smokers at time of recruitment

Y N

154 (64.4) 85 (35.6)*

137 (66.8) 68 (33.2)*

17 (50.0) 17 (50.0)

5 (62.5) 3 (37.5)

Ever smoked

Y N

145 (88.4) 19 (11.6)**

123 (94.6) 7 (5.4)**

22 (64.7) 12 (35.3)

7 (87.5) 1 (12.5)

Smoking at time st of 1 psoriatic manifestation Family history positive

Y N

173 (74.6) 59 (25.4)***

157 (78.9) 42 (21.1)***

16 (48.5) 17 (51.5)***

6 (75.0) 2 (25.0)

Y N

75 (32.5) 156 (67.5)#

71 (36.0) 126 (64.0)#

4 (11.8) 30 (88.2)

1 (14.3) 6 (85.7)#

Additional PsV

Y N

41 (16.6) 206 (83.4)##

41 (19.2) 172 (80.8)##

0 (0.0) 34 (100.0)

1 (12.5) 7 (87.5)

Additional PsA

Y N

59 (24.6) 181 (75.4)###

59 (28.6) 147 (71.4)###

0 (0.0) 34 (100.0)

2 (28.6) 5 (71.4)###

* Unknown status in 12 individuals; ** unknown status in 81 individuals; *** unknown status in 19/ 18/ 1 individuals, respectively; # unknown status in 20/ 20/ 1 individuals, respectively; ## unknown status in 4 individuals; ### unknown status in 11/ 11/ 1 individuals, respectively.

© 2015 The Society for Investigative Dermatology

[numbers indicate absolute counts (percentages)], n.a. = not applicable

34 Estonian PPP

German controls (n=932/937)

Estonian controls (n=117)

European controls (Jordan et al., 2012a) (n=1,609)

ESP controls (n=4,2824,300)*

0 (0)

0 (0)

0 (0)

0 (0)

n.a.

1 (0.01)

/

c.227C>T/ p.Pro76Leu

1 (0.23)

0 (0)

1 (0.05)

0 (0)

n.a.

2 (0.02)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

n.a.

0 (0)

0 (0)

c.338C>T/ p.Ser113Leu

2 (0.46)

1 (1.47)

6 (0.32)

0 (0)

n.a.

27 (0.31)

3 (0.40)

∑ mutant alleles

3 (0.69)

1 (1.47)

7 (0.38)

0 (0)

n.a.

30 (0.35)

3 (0.40)

8,570 (99.65)

755 (99.60)

4 (0.80) ∑ wildtype alleles

431 (99.31)

40 (0.35)

67 (98.53)

1,857 (99.62)

234 (100)

498 (99.20)

n.a. 11,416 (99.65)

c.599G>A/ p.Ser200Asn

5 (1.05)

1 (1.47)

4 (0.22)

3 (1.28)

0 (0)

2 (0.26)

27 (0.84)

c.536G>A/ p.Arg179His

0 (0)

0 (0)

1 (0.0)

0 (0)

1 (0.02)

/

1 (0.03)

c.526G>C/ p.Asp176His

0 (0)

2 (2.94)

2 (0.11)

1 (0.43)

2 (0.02)

0 (0)

2 (0.06)

5 (1.15)

3 (4.41)

7 (0.38)

4 (1.71)

3 (0.04)

2 (0.26)

30 (0.93)

756 (99.74)

3,188 (99.07)

∑ mutant alleles

8 (1.59) CARD14

1000 genomes (n=379)**

c.142C>T/ p.Arg48Trp

c.280G>T/ p.Glu94X IL36RN

217 German PPP

∑ wildtype alleles

429 (98.85)

p-value

Variant/ combination of haplotype

Control groups

0.11

Locus

PPP (n=251)

46 (0.32)

65 (95.59)

1,825 (99.62)

494 (98.41)

230 (98.29)

8,561 (99.96)

14,560 (99.68)

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3.81E-04

Table 2: Association analysis at three genes/ loci.

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PSORS1

GTC/ other ∑ homozygous/ heterozygous carriers of GTC Other/ other

1 (0.46)

1 (2.94)

3 (0.32)

1 (0.85)

n.a.

n.a.

n.a.

38 (17.51)

3 (8.82)

128 (13.66)

19 (16.24)

n.a.

n.a.

n.a.

n.a.

n.a.

43 (17.13) 178 (82.03)

∑ other/ other

0.26

GTC/ GTC haplotype

151 (14.33)

30 (88.24)

806 (86.02)

208 (82.87)

95 (82.91)

n.a. 903 (85.67)

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Journal of Investigative Dermatology

Palmoplantar pustular psoriasis is associated with missense variants in CARD14 in European patients, but not with lossof-function mutations in IL36RN in European patients

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Manuscript ID: Manuscript Type:

Complete List of Authors:

JID-2014-0309.R4 Letter to Editor 20-Apr-2015

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Date Submitted by the Author:

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Mössner, Rotraut; Georg-August-University Göttingen, Department of Dermatology Frambach, Yvonne; Universität zu Lübeck, Department of Dermatology Wilsmann-Theis, Dagmar; University Bonn, Department of Dermatology and Allergy Löhr, Sabine; Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Human Genetics Jacobi, Arnd; Department of Dermatology, University Marburg Weyergraf, Ansgar; Fachklinik Bad Bentheim, Department of Dermatology Müller, Michael; Georg-August-University Göttingen, Department of Occupational, Social and Environmental Medicine Philipp, Sandra; Universitätsmedizin Berlin, Department of Dermatology and Allergy Renner, Regina; University of Erlangen-Nuremberg, Department of Dermatology Traupe, Heiko; University Münster, Department of Dermatology Burkhardt, Harald; Johann Wolfgang Goethe University, Division of Rheumatology and IME-Fraunhofer Project Group Translational Medicine and Pharmacology Kingo, Külli; University of Tartu, Department of Dermatology Koks, Sulev; University of Tartu, Department of Pathophysiology Uebe, Steffen; Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Human Genetics Sticherling, Michael; University of Erlangen-Nuremberg, Department of Dermatology Sticht, Heinrich; Friedrich-Alexander-Universität Erlangen-Nürnberg, Bioinformatics, Institute of Biochemistry Oij, Vincent; University Münster, Department of Dermatology Hüffmeier, Ulrike; Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Human Genetics

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Key Words:

Palmoplantar pustular psoriasis, IL36RN, CARD14, PSORS1, psoriasis vulgaris

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Journal of Investigative Dermatology

© 2015 The Society for Investigative Dermatology

Journal of Investigative Dermatology

Palmoplantar pustular psoriasis is associated with missense variants in CARD14, but not with loss-of-function mutations in IL36RN in European patients

Rotraut Mössner 1, Yvonne Frambach 2,3, Dagmar Wilsmann-Theis 4, Sabine Löhr 5, Arnd Jacobi 6, Ansgar Weyergraf 7, Michael Müller 8, Sandra Philipp 9, Regina Renner 10, Heiko Traupe 11, Harald Burkhardt 12, Külli Kingo 13, Sulev Kõks 14, Steffen Uebe 5, Michael Sticherling 10, Heinrich Sticht 15, Vinzenz Oji 11, Ulrike Hüffmeier 5

1 Department of Dermatology, Georg-August-University Göttingen, Göttingen, Germany

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2 Universität zu Lübeck, Department of Dermatology, Lübeck, Germany 3 Hanse-Klinik, Lübeck, Germany

4 Department of Dermatology and Allergy, University Bonn, Bonn, Germany 5 Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

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6 Institute for Health Services Research in Dermatology and Nursing, University Medical Center Hamburg-Eppendorf, Hamburg and Department of Dermatology, University Marburg, Marburg, Germany

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7 Department of Dermatology, Fachklinik Bad Bentheim, Bad Bentheim, Germany

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8 Institute of Occcupational, Social and Environmental Medicine, Georg-August-University Göttingen, Göttingen, Germany 9 Department of Dermatology and Allergy, Universitätsmedizin Berlin, Berlin, Germany 10 Department of Erlangen, Germany

Dermatology,

Friedrich-Alexander-Universität

On

Erlangen-Nürnberg,

11 Department of Dermatology, University Münster, Münster, Germany 12 Division of Rheumatology and IME-Fraunhofer Project Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

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13 Dermatology Clinic, Clinics of Tartu University and Department of Dermatology, University of Tartu, Tartu, Estonia 14 Department of Pathophysiology, University of Tartu, Tartu, Estonia 15 Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Germany

Corresponding author: PD Dr. Ulrike Hüffmeier, Institute of Human Genetics, FriedrichAlexander-Universität Erlangen-Nürnberg, Schwabachanlage 10, 90154 Erlangen, Germany, email: [email protected], Phone: +49 9131 85 24192, Fax: +49 9131 85 23232

© 2015 The Society for Investigative Dermatology

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Abbreviations: PPP = palmoplantar pustular psoriasis, GPP = generalized pustular psoriasis, PsV = psoriasis vulgaris, PsA = psoriatic arthritis

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Mössner et al.: PPP and missense variants in CARD14

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Journal of Investigative Dermatology

To the editor Psoriasis is a complex genetic disease, meaning that a combination of genetic and environmental risk factors leads to disease manifestation. The most common cutaneous manifestation is psoriasis vulgaris (PsV) which is strongly associated with an HLA-C risk allele located at psoriasis susceptibility locus 1 (PSORS1). This locus has a greater impact on PsV, while there are many other genetic risk factors, but most of these contribute far less. Only in some families and single patients suffering from PsV as well as in patients with a severe pustular variant of psoriasis, named generalized pustular psoriasis (GPP), few other

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major genetic risk factors responsible for those disease manifestations were identified. These include missense variants in CARD14 and loss-of-function mutations in IL36RN described to be responsible for PsV and GPP or GPP, respectively (Jordan et al., 2012a; Jordan et al.,

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2012b; Marrakchi et al., 2011; Onoufriadis et al., 2011). Palmoplantar pustular psoriasis, also termed palmoplantar pustulosis (PPP), is characterized

vi

by sterile pustules on the palms and soles. It is more frequent in women and has stronger

ew

association with tobacco smoking than PsV or psoriatic arthritis (PsA) (O'Doherty and MacIntyre, 1985). While no randomized controlled studies with biologics licensed for PsV

On

have been published, conventional therapy is similar to PsV, although there are differences in therapeutic outcome (De Mozzi et al., 2012; Marsland et al., 2006). Little is known about genetic risk factors of PPP; however, an association of PPP with missense mutations in

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IL36RN has been described recently (Setta-Kaffetzi et al., 2013). There is an ongoing discussion on whether PPP is a pustular variant of psoriasis or a distinct disease entity (Ammoury et al., 2008; Brunasso et al., 2013). In order to characterize PPP genetically, we recruited a cohort of 251 PPP patients and screened them for the major genetic risk factor for PsV at PSORS1 as well as for the two genes IL36RN and CARD14 as described previously (Huffmeier et al., 2009; Korber et al., 2013). Approval for the studies was obtained through the ethical committees of the responsible universities. Individuals gave their written informed consent. European Mössner et al.: PPP and missense variants in CARD14

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Americans from publicly available databases, from a previous publication (Jordan et al., 2012a) and control individuals from a German cohort (Huffmeier et al., 2009) served as control groups (for slight variations in control individuals, please see Supplements). The majority of the 251 PPP patients was female (78.1%) and 88.4% of recruited patients had a history of smoking, with 75% smoking at the time of their first psoriatic manifestation (Table 1, Supplements). The percentages of patients with concomitant PsV and a positive family history for psoriatic manifestations were 16.6% and 32.5%, respectively, and 24.6% of PPP patients reported to have been diagnosed with PsA.

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Four of the 251 PPP patients were identified to carry previously identified IL36RN mutations by Sanger-sequencing (Table 2): one carried c.227C>T/ p.Pro76Leu in a heterozygous form and three further patients were heterozygous carriers of c.338C>T/ p.Ser113Leu, summing

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up to a carrier rate of 2.1%. In addition, a further patient was heterozygous for c.17C>T/ p.Ala6Val, a missense variant in IL36RN. To the best of our knowledge, this variant has not

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been observed before. Molecular modeling revealed that valine at the 6th amino acid position

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causes some minor local structural changes, but due to a neighboring disulfide-bridge formed by Cys8, the global structure of the N-terminus can be stabilized. The effect at protein level is probably marginal and thus the variant was excluded from further association

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analyses. In comparison to different control groups (Table 2), we observed no increased frequency of mutant alleles in IL36RN and therefore no evidence for association. Previously,

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association of PPP with IL36RN mutations has been described with an effect size (odds ratio (OR)) of 12.3 [5.8-25.8] (Setta-Kaffetzi et al., 2013). Our study had 80% power to detect effects sizes lower than the lower limit of the 95% confidence interval in that study. With our cohort we had >99.999% power to detect an association of PPP with IL36RN mutant alleles based on allele frequency and effect size reported previously (Setta-Kaffetzi et al., 2013). Sequencing of the first three coding exons of CARD14 in 251 PPP patients revealed several interesting coding variants: Six heterozygous carriers of c.599G>A/ p.Ser200Asn, and two heterozygous carriers of c.526G>C/ p.Asp176His were identified (Table 2). In two different Mössner et al.: PPP and missense variants in CARD14

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groups of 1,049 controls genotyped in-house, we identified additionally c.536G>A/ p.Arg179His in heterozygous state in one control individual and 10 heterozygous carriers of c.599G>A/ p.Ser200Asn or c.526G>C/ p.Asp176His. These missense variants have been previously described to be involved in PsV and GPP (Jordan et al., 2012a; Korber et al., 2013; Sugiura et al., 2014). In addition, we identified the missense variant c.589G>A/ p.Glu197Lys exclusively in three German controls, previously neither reported nor functionally tested. Therefore, we excluded this variant from further analyses. With regard to the three functionally tested variants and in comparison to additional publicly available data

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at the exome variant server (http://evs.gs.washington.edu/EVS/), within the 1000 genomes project (Genomes Project et al., 2010) (http://www.1000genomes.org/) and a previous control cohort of 1,609 control individuals (Jordan et al., 2012a), we observed a highly

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significant association (p = 3.81E-04) and a corresponding OR of 5.13 [2.08-11.04]. Of note, the effect size is considerably higher than the effect sizes described in PsV (Jordan et al.,

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2012a).

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Of the eight PPP patients carrying the three missense variants in CARD14 (Table 3, last column), one patient (12.5%) had concomitant PsV and PsA, another patient had concomitant PsA only. Among the PPP patients, neither the frequencies of PsV and PsA) nor

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the frequency of patients who had ever smoked or had a history of smoking differed between carriers and non-carriers of the three CARD14 missense variants. Both the percentage of

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male patients carrying a CARD14 variant and the percentage of patients with negative family history for a psoriatic manifestation were higher compared to PPP patients without CARD14 missense variants. However, these results have to be interpreted with caution due to the small size of the groups. With regard to the PSORS1 risk allele, we observed no significant differences in carrier frequencies between PPP patients and healthy controls (Table 2). We had 80% power to detect an OR of at least 1.73 given the observed allele frequency. This effect size is considerably lower than the one observed previously in our large cohorts of PsV and PsA

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patients: OR = 8.1 [6.5–10.1] for PsV and OR = 4.5 [3.6–5.7] for PsA. Our negative association finding is in concordance to previous studies (Asumalahti et al., 2003; Fan et al., 2007; Karvonen et al., 1976). Overall, our investigations suggest an involvement of missense variants in CARD14 in the pathogenesis of PPP, but do not support a role for IL36RN mutations. These results suggest that genetic risk factors in PPP overlap with PsV and GPP, while we hypothesize that there will be additional, so far unknown, genetic risk factors probably restricted to PPP that are responsible for the typical, locally restricted palmoplantar pustular manifestation.

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Conflict of Interest

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The authors declare that there is no conflict of interest.

Acknowledgements

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We acknowledge all patients and control probands for participation in this study making it possible. We thank Daniela Hemmeter and Anne Gerschütz for excellent technical assistance. Parts of this study were supported by two different grants to U.H.: a) from the

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German Research Foundation (DFG 2163/1-1) and b) from the ELAN funding of the Friedrich-Alexander-Universität

Erlangen-Nürnberg

(“Erlanger

Leistungsbezogene

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Anschubfinanzierung und Nachwuchsförderung”, ELAN-13-03-09-1-UH). The contribution of H.B. was supported by the Federal State of Hesse (LOEWE-Project: Fraunhofer IME-ProjectGroup Translational Medicine and Pharmacology, Goethe University Frankfurt).

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References Ammoury A, El Sayed F, Dhaybi R, et al. (2008) Palmoplantar pustulosis should not be considered as a variant of psoriasis. Journal of the European Academy of Dermatology and Venereology : JEADV 22:392-3. Asumalahti K, Ameen M, Suomela S, et al. (2003) Genetic analysis of PSORS1 distinguishes guttate psoriasis and palmoplantar pustulosis. The Journal of investigative dermatology 120:627-32. Brunasso AM, Puntoni M, Aberer W, et al. (2013) Clinical and epidemiological comparison of patients affected by palmoplantar plaque psoriasis and palmoplantar pustulosis: a case series study. The British journal of dermatology 168:1243-51. De Mozzi P, Johnston GA, Alexandroff AB (2012) Psoriasis: an evidence-based update. Report of the 9th evidenced based update meeting, 12 May 2011, Loughborough, UK. The British journal of dermatology 166:252-60.

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Fan X, Yang S, Sun LD, et al. (2007) Comparison of clinical features of HLA-Cw*0602positive and -negative psoriasis patients in a Han Chinese population. Acta dermatovenereologica 87:335-40.

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Genomes Project C, Abecasis GR, Altshuler D, et al. (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061-73. Huffmeier U, Lascorz J, Bohm B, et al. (2009) Genetic variants of the IL-23R pathway: association with psoriatic arthritis and psoriasis vulgaris, but no specific risk factor for arthritis. The Journal of investigative dermatology 129:355-8.

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Jordan CT, Cao L, Roberson ED, et al. (2012a) Rare and common variants in CARD14, encoding an epidermal regulator of NF-kappaB, in psoriasis. American journal of human genetics 90:796-808. Jordan CT, Cao L, Roberson ED, et al. (2012b) PSORS2 is due to mutations in CARD14. American journal of human genetics 90:784-95.

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Karvonen J, Tilikainen A, Lassus A (1976) HLA antigens in psoriasis. A family study. Annals of clinical research 8:298-304.

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Korber A, Mossner R, Renner R, et al. (2013) Mutations in IL36RN in patients with generalized pustular psoriasis. The Journal of investigative dermatology 133:2634-7. Marrakchi S, Guigue P, Renshaw BR, et al. (2011) Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis. The New England journal of medicine 365:620-8. Marsland AM, Chalmers RJ, Hollis S, et al. (2006) Interventions for chronic palmoplantar pustulosis. Cochrane database of systematic reviews:CD001433. O'Doherty CJ, MacIntyre C (1985) Palmoplantar pustulosis and smoking. British medical journal 291:861-4. Onoufriadis A, Simpson MA, Pink AE, et al. (2011) Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. American journal of human genetics 89:432-7. Mössner et al.: PPP and missense variants in CARD14

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Setta-Kaffetzi N, Navarini AA, Patel VM, et al. (2013) Rare pathogenic variants in IL36RN underlie a spectrum of psoriasis-associated pustular phenotypes. The Journal of investigative dermatology 133:1366-9. Sugiura K, Muto M, Akiyama M (2014) CARD14 c.526G>C (p.Asp176His) is a significant risk factor for generalized pustular psoriasis with psoriasis vulgaris in the Japanese cohort. The Journal of investigative dermatology 134:1755-7.

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Table 1: Clinical characteristics of PPP patients Numbers indicate absolute counts (and percentages). PPP = PPP patients, Y = yes, N = no.

Category

Subcategory

217 German PPP

34 Estonian PPP

8 PPP carrying CARD14 variants

51.1±12.9

50.6±13.1

54.7±11.1

54.4±14.5

41.8±13.6

41.4±12.6

49.0±10.2

45.7±16.9

Average age at recruitment Average age at psoriatic 1st manifestation Gender male/ female

male female

55 (21.9) 196 (78.1)

47 (21.7) 170 (78.3)

8 (23.5) 26 (76.5)

3 (37.5) 5 (62.5)

Smokers at time of recruitment

Y N

154 (64.4) 85 (35.6)*

137 (66.8) 68 (33.2)*

17 (50.0) 17 (50.0)

5 (62.5) 3 (37.5)

Y N

145 (88.4) 19 (11.6)**

123 (94.6) 7 (5.4)**

22 (64.7) 12 (35.3)

7 (87.5) 1 (12.5)

Smoking at time st of 1 psoriatic manifestation Family history positive

Y N

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251 PPP

173 (74.6) 59 (25.4)***

157 (78.9) 42 (21.1)***

16 (48.5) 17 (51.5)***

6 (75.0) 2 (25.0)

75 (32.5) 156 (67.5)#

71 (36.0) 126 (64.0)#

4 (11.8) 30 (88.2)

1 (14.3) 6 (85.7)#

Additional PsV

Y N

41 (16.6) 206 (83.4)##

41 (19.2) 172 (80.8)##

0 (0.0) 34 (100.0)

1 (12.5) 7 (87.5)

Additional PsA

Y N

59 (24.6) 181 (75.4)###

59 (28.6) 147 (71.4)###

0 (0.0) 34 (100.0)

2 (28.6) 5 (71.4)###

Ever smoked

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* Unknown status in 12 individuals; ** unknown status in 81 individuals; *** unknown status in 19/ 18/ 1 individuals, respectively; # unknown status in 20/ 20/ 1 individuals, respectively; ## unknown status in

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4 individuals; ### unknown status in 11/ 11/ 1 individuals, respectively.

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[numbers indicate absolute counts (percentages)], n.a. = not applicable

c.142C>T/ p.Arg48Trp c.227C>T/ p.Pro76Leu

IL36RN

c.280G>T/ p.Glu94X

217 German PPP

34 Estonian PPP

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Estonian controls (n=117)

European controls (Jordan et al., 2012a) (n=1,609)

ESP controls (n=4,2824,300)*

0 (0)

0 (0)

0 (0)

n.a.

1 (0.01)

/

1 (0.23)

0 (0)

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1 (0.05)

0 (0)

n.a.

2 (0.02)

0 (0)

0 (0)

0 (0)

0 (0)

n.a.

0 (0)

0 (0)

6 (0.32)

0 (0)

n.a.

27 (0.31)

3 (0.40)

7 (0.38)

0 (0)

n.a.

30 (0.35)

3 (0.40)

8,570 (99.65)

755 (99.60)

2 (0.26)

27 (0.84)

1 (0.02)

/

1 (0.03)

2 (0.02)

0 (0)

2 (0.06)

3 (0.04)

2 (0.26)

30 (0.93)

756 (99.74)

3,188 (99.07)

0 (0)

2 (0.46)

∑ mutant alleles

3 (0.69)

1 (1.47)

1 (1.47) 4 (0.80)

431 (99.31)

67 (98.53)

498 (99.20)

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1,857 (99.62)

40 (0.35)

234 (100)

5 (1.05)

1 (1.47)

4 (0.22)

3 (1.28)

c.536G>A/ p.Arg179His

0 (0)

0 (0)

1 (0.0)

0 (0)

c.526G>C/ p.Asp176His

0 (0)

2 (2.94)

2 (0.11)

1 (0.43)

5 (1.15)

3 (4.41)

7 (0.38)

4 (1.71)

8 (1.59) ∑ wildtype alleles

429 (98.85)

n.a.

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c.599G>A/ p.Ser200Asn

∑ mutant alleles

1000 genomes (n=379)**

0 (0)

c.338C>T/ p.Ser113Leu

∑ wildtype alleles

German controls (n=932/937)

11,416 (99.65) 0 (0)

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46 (0.32)

65 (95.59)

1,825 (99.62)

494 (98.41)

230 (98.29)

8,561 (99.96)

14,560 (99.68)

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p-value

Variant/ combination of haplotype

Control groups

0.11

Locus

PPP (n=251)

3.81E-04

Table 2: Association analysis at three genes/ loci.

CARD14

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GTC/ other ∑ homozygous/ heterozygous carriers of GTC Other/ other ∑ other/ other

1 (0.46)

1 (2.94)

3 (0.32)

1 (0.85)

n.a.

n.a.

n.a.

38 (17.51)

3 (8.82)

128 (13.66)

19 (16.24)

n.a.

n.a.

n.a.

n.a.

n.a.

43 (17.13)

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178 (82.03)

0.26

GTC/ GTC haplotype

PSORS1

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151 (14.33)

30 (88.24)

806 (86.02)

95 (82.91)

rR

n.a. 903 (85.67)

208 (82.87)

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Palmoplantar pustular psoriasis is associated to missense variants in CARD14 in European patients

Rotraut Mössner 1, Yvonne Frambach 2,3, Dagmar Wilsmann-Theis 4, Sabine Löhr 5, Arnd Jacobi 6, Ansgar Weyergraf 7, Michael Müller 8, Sandra Philipp 9, Regina Renner 10, Heiko Traupe 11, Harald Burkhardt 12, Külli Kingo 13, Sulev Kõks 14, Steffen Uebe 5, Michael Sticherling 10, Heinrich Sticht 15, Vinzenz Oji 11, Ulrike Hüffmeier 5

1 Department of Dermatology, Georg-August-University Göttingen, Göttingen, Germany

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2 Universität zu Lübeck, Department of Dermatology, Lübeck, Germany 3 Hanse-Klinik, Lübeck, Germany

4 Department of Dermatology and Allergy, University Bonn, Bonn, Germany 5 Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

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6 Institute for Health Services Research in Dermatology and Nursing, University Medical Center Hamburg-Eppendorf, Hamburg and Department of Dermatology, University Marburg, Marburg, Germany

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7 Department of Dermatology, Fachklinik Bad Bentheim, Bad Bentheim, Germany

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8 Institute of Occcupational, Social and Environmental Medicine, Georg-August-University Göttingen, Göttingen, Germany 9 Department of Dermatology and Allergy, Universitätsmedizin Berlin, Berlin, Germany 10 Department of Erlangen, Germany

Dermatology,

Friedrich-Alexander-Universität

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Erlangen-Nürnberg,

11 Department of Dermatology, University Münster, Münster, Germany 12 Division of Rheumatology and IME-Fraunhofer Project Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

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13 Dermatology Clinic, Clinics of Tartu University and Department of Dermatology, University of Tartu, Tartu, Estonia 14 Department of Pathophysiology, University of Tartu, Tartu, Estonia 15 Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Germany

© 2015 The Society for Investigative Dermatology

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Detailed materials and methods 217 European patients with PPP diagnosed by board certified dermatologists were recruited at six different university hospitals and one rehabilitation hospital in Germany. 34 Caucasian patients with PPP without concomitant psoriasis vulgaris and 117 healthy controls were enrolled at the Department of Dermatology, University of Tartu, Estonia. They represent the majority of the patient collectives described before by Mössner et al. (2005). They had neither symptoms of inflammatory joint disease nor a personal history of psoriatic arthritis, and recruiting doctors did not identify any joint deformities. Detailed clinical and anamnestic data of patients were collected and documented (Table 3). In the Estonian control group, age at recruitment was 35.2 ± 14.2 years, and 74 individuals (63.2%) were female. Approval for

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the studies was obtained through the ethical committees of the responsible universities. Individuals gave their written informed consent. The investigations were conducted according to the Declaration of Helsinki principles. All PPP patients and 117 Estonian control individuals were screened for mutations by Sanger

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sequencing for the four coding exons of IL36RN and the first three coding exons of CARD14 (exons 2-4 according to NM_024110). In addition, a deletion/ duplication analysis of coding exons of the IL36RN gene and the first two coding exons of the CARD14 gene (exons 2-3)

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was performed with MLPA. We observed no carrier of a deletion of IL36RN or a partial deletion of CARD14. A more detailed description of methods has been published recently

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(Korber et al., 2013). In addition, the PSORS1 risk allele was analyzed in PPP patients as described before (Huffmeier et al., 2009). Non-synonymous variants and their consequences on protein structure/ function were modeled as described recently (Korber et al., 2013).

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We designed probes and primers for TaqMan based assays to determine carriers of any of the four different IL36RN mutations exclusively and previously identified in 19 GPP patients of German origin (Korber et al., 2013). By using those assays, we screened 932 healthy

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control individuals from Germany; those cohorts have been analyzed in very similar compositions for other projects (Bohm et al., 2012; Huffmeier et al., 2010). DNAs with insufficient genotyping quality or genotyping failure were sequenced by Sanger method as described before (Korber et al., 2013). In addition, we sequenced the 3rd coding exon of CARD14 (exon 4) in 916 German control individuals. Fisher’s Exact test, as implemented in R (version 2.15.3) (R-Core-Team, 2013), was used to check for significant differences in allele frequency between case groups of one psoriatic disease entity and controls. Power at the 0.05 significance level was determined using the power.fisher.test function (package statmod) (Smyth, 1989) with 100,000 simulations. For association analysis of PPP at PSORS1, we compared carriers - homozygous or heterozygous - of the PSORS1 risk haplotype GTC to non-carriers by using a Chi-Square

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test of independence. As a control group, we used the largely overlapping group of 937 individuals described previously (Huffmeier et al., 2009) as well as an additional, newly genotyped group of Estonian controls.  

 

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References Bohm B, Burkhardt H, Uebe S, et al. (2012) Identification of low-frequency TRAF3IP2 coding variants in psoriatic arthritis patients and functional characterization. Arthritis research & therapy 14:R84. Huffmeier U, Lascorz J, Bohm B, et al. (2009) Genetic variants of the IL-23R pathway: association with psoriatic arthritis and psoriasis vulgaris, but no specific risk factor for arthritis. The Journal of investigative dermatology 129:355-8. Huffmeier U, Uebe S, Ekici AB, et al. (2010) Common variants at TRAF3IP2 are associated with susceptibility to psoriatic arthritis and psoriasis. Nature genetics 42:996-9. Korber A, Mossner R, Renner R, et al. (2013) Mutations in IL36RN in patients with generalized pustular psoriasis. The Journal of investigative dermatology 133:2634-7.

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Mossner R, Kingo K, Kleensang A, et al. (2005) Association of TNF -238 and -308 promoter polymorphisms with psoriasis vulgaris and psoriatic arthritis but not with pustulosis palmoplantaris. The Journal of investigative dermatology 124:282-4.

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R-Core-Team (2013) R: A language and environment for statistical computing. . (R Foundation for Statistical Computing, Vienna, Austria.

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Smyth GK (1989) Generalized linear models with varying dispersion. J R Statist Soc 51:4761.

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Palmoplantar Pustular Psoriasis Is Associated with Missense Variants in CARD14, but Not with Loss-of-Function Mutations in IL36RN in European Patients.

Palmoplantar Pustular Psoriasis Is Associated with Missense Variants in CARD14, but Not with Loss-of-Function Mutations in IL36RN in European Patients. - PDF Download Free
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