Loss of IL36RN Function Does Not Confer Susceptibility to Psoriasis Vulgaris
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Loss of IL36RN Function Does Not Confer Susceptibility to Psoriasis Vulgaris Journal of Investigative Dermatology advance online publication, 18 July 2013; doi:10.1038/jid.2013.285 TO THE EDITOR Recessive mutations of the gene encod- ing the interleukin-36 receptor antago- nist (IL36RN) have been associated with generalized pustular psoriasis, palmar- plantar pustulosis, and acrodermatitis continua of Hallopeau (Marrakchi et al., 2011; Onoufriadis et al., 2011; Setta-Kaffetzi et al., 2013). As patients suffering from these pustular conditions often present with concomitant psoriasis vulgaris (PV), it has been proposed that IL36RN deficiency may also contri- bute to PV susceptibility (Marrakchi et al., 2011). This hypothesis is sup- ported by the observation that mice lacking il36rn show exacerbated symp- toms of imiquimod-induced psoriasiform dermatitis and enhanced infiltration of inflammatory cells in the dermis and the epidermis (Tortola et al., 2012). The elevated expression of IL-36 cytokines in psoriatic skin (Carrier et al., 2011; Johnston et al., 2011) is also consistent with the notion that abnormal IL-36 signaling has an important role in the establishment of cutaneous inflamma- tion (Supplementary Figure 1 online). On the basis of the above findings, it has recently been suggested that IL-36 blockade could be an innova- tive approach for the treatment of PV (Tortola et al., 2012). However, the pathogenic role of IL-36 cytokines has mostly been investigated in the context of animal models (Blumberg et al., 2010; Carrier et al., 2011; Tortola et al., 2012), and the notion that IL36RN deficiency contributes to the onset of human PV has yet to be demonstrated. Here, we have explored this hypothesis by investigating whether IL36RN loss-of-function alleles are associated with PV. We reasoned that the segregation of deleterious sequence variants would result in familial recurrence of PV and ascertained 349 unrelated patients with a positive family history of the disease (174 male and 175 female individuals; see also Table 1). We also examined 14 independent cases of erythrodermic psoriasis (10 male and 4 female individuals), based on the hypothesis that penetrant mutations would more likely result in a severe clinical presentation. All patients were recruited through St John’s Institute of Dermatology and Glasgow Western Infirmary. The study was conducted in accordance with the declaration of Helsinki, and ethical approval was obtained from Guy’s and St Thomas Local Research Ethics committee. All participants granted their written informed consent. The four IL36RN coding exons and proximal promoter region were sequen- ced using primers and cycling condi- tions described elsewhere (Onoufriadis et al., 2011; Setta-Kaffetzi et al., 2013). Nucleotide changes were iden- tified using the Sequencher software (Genecodes). Sanger sequencing identified six com- mon polymorphisms with minor allele frequencies exceeding 5%. None of these changes resulted in an amino- acid substitution and none of the vari- ants was significantly overrepresented in cases compared with controls (Supple- mentary Table 1 online). To exclude the possibility that these findings may be due to lack of statistical power, we reexamined the IL36RN markers ana- lyzed in a previous genome-wide asso- ciation study of 2,600 psoriatic cases and 5,700 controls (Strange et al., 2010). By integrating experimental and imputed data (see Supplementary Methods online), we obtained geno- types for 130 single-nucleotide poly- morphisms spanning the IL36RN locus and 10 kb of flanking regions. None of these markers generated a P-value o0.05, indicating that common IL36RN variants are unlikely to confer significant increases in disease risk. None of the examined patients har- bored recessive alleles with pathogenic potential (i.e., homozygous or com- pound heterozygous changes resulting in non-synonymous substitutions or protein truncation). Three affected indi- viduals were heterozygous for rare non- synonymous substitutions. Two carried a known p.Ser113Leu (rs144478519) change and one previously unreported p.Thr77Ile variant. The latter substitu- tion was classified as benign by the SIFT (Kumar et al., 2009) and PolyPhen-2 (Adzhubei et al., 2010) pathogenicity prediction tools and therefore was not analyzed any further. Conversely, the p.Ser113Leu allele is known to be damaging, as it has been repeatedly detected in patients with pustular psoriasis (Onoufriadis et al., 2011; Setta-Kaffetzi et al., 2013). As data pre- viously generated by our group sugges- ted that this mutation may have a pathogenic effect even in the hetero- zygous state (Navarini et al., 2013; Setta-Kaffetzi et al., 2013), we investi- gated its segregation in the families of the two critical patients. In both cases, we found that the p.Ser113Leu substitution was not co-inherited with the disease (Supplementary Figure 2 online). We also observed that the frequency of p.Ser113Leu in our patient resource (2/726 chromosomes or 0.27%) was comparable to that seen in 2,222 exomes sequenced in-house (15/4,444 chromosomes or 0.34%). Thus, the p.Ser113Leu LETTER TO THE EDITOR Accepted article preview online 21 June 2013 Abbreviations: IL36RN, interleukin-36 receptor antagonist gene; PV, psoriasis vulgaris & 2013 The Society for Investigative Dermatology www.jidonline.org 1
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Transcript of Loss of IL36RN Function Does Not Confer Susceptibility to Psoriasis Vulgaris
Loss of IL36RN Function Does Not Confer Susceptibilityto Psoriasis VulgarisJournal of Investigative Dermatology advance online publication, 18 July 2013; doi:10.1038/jid.2013.285
TO THE EDITORRecessive mutations of the gene encod-ing the interleukin-36 receptor antago-nist (IL36RN) have been associated withgeneralized pustular psoriasis, palmar-plantar pustulosis, and acrodermatitiscontinua of Hallopeau (Marrakchiet al., 2011; Onoufriadis et al., 2011;Setta-Kaffetzi et al., 2013). As patientssuffering from these pustular conditionsoften present with concomitant psoriasisvulgaris (PV), it has been proposed thatIL36RN deficiency may also contri-bute to PV susceptibility (Marrakchiet al., 2011). This hypothesis is sup-ported by the observation that micelacking il36rn show exacerbated symp-toms of imiquimod-induced psoriasiformdermatitis and enhanced infiltration ofinflammatory cells in the dermis andthe epidermis (Tortola et al., 2012). Theelevated expression of IL-36 cytokinesin psoriatic skin (Carrier et al., 2011;Johnston et al., 2011) is also consistentwith the notion that abnormal IL-36signaling has an important role in theestablishment of cutaneous inflamma-tion (Supplementary Figure 1 online).
On the basis of the above findings,it has recently been suggested thatIL-36 blockade could be an innova-tive approach for the treatment of PV(Tortola et al., 2012). However, thepathogenic role of IL-36 cytokineshas mostly been investigated in thecontext of animal models (Blumberget al., 2010; Carrier et al., 2011;Tortola et al., 2012), and the notionthat IL36RN deficiency contributes tothe onset of human PV has yet to bedemonstrated. Here, we have exploredthis hypothesis by investigating whetherIL36RN loss-of-function alleles areassociated with PV.
We reasoned that the segregation ofdeleterious sequence variants wouldresult in familial recurrence of PVand ascertained 349 unrelated patientswith a positive family history of thedisease (174 male and 175 femaleindividuals; see also Table 1). We alsoexamined 14 independent cases oferythrodermic psoriasis (10 male and4 female individuals), based on thehypothesis that penetrant mutationswould more likely result in a severeclinical presentation. All patients wererecruited through St John’s Instituteof Dermatology and Glasgow WesternInfirmary. The study was conductedin accordance with the declarationof Helsinki, and ethical approvalwas obtained from Guy’s andSt Thomas Local Research Ethicscommittee. All participants grantedtheir written informed consent.
The four IL36RN coding exons andproximal promoter region were sequen-ced using primers and cycling condi-tions described elsewhere (Onoufriadiset al., 2011; Setta-Kaffetzi et al.,2013). Nucleotide changes were iden-tified using the Sequencher software(Genecodes).
Sanger sequencing identified six com-mon polymorphisms with minor allelefrequencies exceeding 5%. None ofthese changes resulted in an amino-acid substitution and none of the vari-ants was significantly overrepresented incases compared with controls (Supple-mentary Table 1 online). To exclude thepossibility that these findings may bedue to lack of statistical power, wereexamined the IL36RN markers ana-lyzed in a previous genome-wide asso-ciation study of 2,600 psoriatic casesand 5,700 controls (Strange et al.,
2010). By integrating experimental andimputed data (see SupplementaryMethods online), we obtained geno-types for 130 single-nucleotide poly-morphisms spanning the IL36RN locusand 10 kb of flanking regions. Noneof these markers generated a P-valueo0.05, indicating that common IL36RNvariants are unlikely to confer significantincreases in disease risk.
None of the examined patients har-bored recessive alleles with pathogenicpotential (i.e., homozygous or com-pound heterozygous changes resultingin non-synonymous substitutions orprotein truncation). Three affected indi-viduals were heterozygous for rare non-synonymous substitutions. Two carrieda known p.Ser113Leu (rs144478519)change and one previously unreportedp.Thr77Ile variant. The latter substitu-tion was classified as benign by the SIFT(Kumar et al., 2009) and PolyPhen-2(Adzhubei et al., 2010) pathogenicityprediction tools and therefore was notanalyzed any further. Conversely, thep.Ser113Leu allele is known to bedamaging, as it has been repeatedlydetected in patients with pustularpsoriasis (Onoufriadis et al., 2011;Setta-Kaffetzi et al., 2013). As data pre-viously generated by our group sugges-ted that this mutation may have apathogenic effect even in the hetero-zygous state (Navarini et al., 2013;Setta-Kaffetzi et al., 2013), we investi-gated its segregation in the familiesof the two critical patients. In bothcases, we found that the p.Ser113Leusubstitution was not co-inherited withthe disease (Supplementary Figure 2online). We also observed that thefrequency of p.Ser113Leu in ourpatient resource (2/726 chromosomesor 0.27%) was comparable to thatseen in 2,222 exomes sequencedin-house (15/4,444 chromosomesor 0.34%). Thus, the p.Ser113Leu
LETTER TO THE EDITOR
Accepted article preview online 21 June 2013
Abbreviations: IL36RN, interleukin-36 receptor antagonist gene; PV, psoriasis vulgaris
& 2013 The Society for Investigative Dermatology www.jidonline.org 1
change is unlikely to contribute to PVsusceptibility.
Data generated in animal modelsindicate that IL-36 cytokines are likelyto have a pathogenic role in theestablishment of skin inflammation.Transgenic mice overexpressing il36ain basal keratinocytes present with apsoriasiform phenotype (IL-23-depen-dent hyperkeratosis accompanied byinfiltration of neutrophils and lympho-cytes into the dermis) that is exacerbatedby il36rn knockout (Blumberg et al.,2007). It has also been demonstratedthat IL-36R blockade resolves theinflammatory changes of human psoria-tic skin transplanted onto immune-deficient mice (Blumberg et al., 2010),whereas loss of il36rn exacerbatesthe pathology of imiquimod-inducedpsoriasiform dermatitis (Tortola et al.,2012).
Despite the interest generated bythese findings, the effect of IL36RNdeficiency on the establishment of PVhas not been investigated. Here, weaddressed this issue through the geneticanalysis of an extended clinical resourceincluding 363 patients affected by famil-ial and/or severe disease. We found thatnone of the examined cases harboredrecessive IL36RN mutations. Althoughtwo patients were heterozygous for thepathogenic p.Ser113Leu mutation, thischange did not segregate with the dis-ease in the relevant families and was notoverrepresented among case chromo-somes. Our patient resource was suffi-ciently powered to uncover variantsoccurring at a frequency 40.25% anddetect associations with alleles of mode-rate to strong effect (SupplementaryFigure 3 online). Although the preva-lence and the odds ratios documentedfor the p.Ser113Leu allele are within
these parameters (Navarini et al., 2013;Setta-Kaffetzi et al., 2013), our analysismight have missed the pathogeniccontribution of rarer alleles of modesteffect. Nonetheless, it is reasonable toconclude that in the British populationloss of IL36RN activity is not associatedwith a significant increase of PV risk.As our study was focused on IL36RN,we cannot exclude the possibility thatmutations in functionally related genes(e.g., IL36A, IL36B, and IL36G) may beassociated with the disease. Thus, ourfindings warrant further genetic investi-gations into the role of IL-36 cytokinesin human PV.
CONFLICT OF INTERESTThe authors state no conflict of interest.
ACKNOWLEDGMENTSWe acknowledge support from the Department ofHealth via the NIHR comprehensive BiomedicalResearch Centre award to GSTT NHS FoundationTrust in partnership with King’s College Londonand KCH NHS Foundation Trust. This work wassupported by the National Psoriasis Foundation,USA (Discovery Grant to FC) and theMedical Research Council (grant G0601387 toRCT and JNB). DB’s PhD studentship is funded bythe Psoriasis Association. SKM is supported by theNIHR through the Academic Clinical Fellowshipscheme.
Dorottya M. Berki1, Satveer K. Mahil1,A. David Burden2,Richard C. Trembath1,3,Catherine H. Smith1,Francesca Capon1,4 andJonathan N. Barker1,4
1Division of Genetics and Molecular Medicine,King’s College London, London, UK;2Department of Dermatology, University ofGlasgow, Glasgow, UK and 3Queen Mary,University of London, Barts and The LondonSchool of Medicine and Dentistry, London, UK4These authors contributed equally to this workE-mail: [email protected] [email protected]
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the onlineversion of the paper at http://www.nature.com/jid
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Table 1. Disease recurrence in the familial PV resource
Number of affected individuals in the pedigree
45 5 4 3 2 Total
No. of families 15 11 33 121 169 349
Abbreviation: PV, psoriasis vulgaris.
DM Berki et al.Loss of IL36RN Function and Psoriasis Vulgaris
2 Journal of Investigative Dermatology
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