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without psoriasis vulgaris
Journal of Dermatological Science 76 (2014) 132–138
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Mutation analysis of the IL36RN gene in Chinese patients with generalized pustular psoriasis with/without psoriasis vulgaris Xiuyan Li a,b,e, Mingfei Chen a,b, Xi’an Fu a,b, Qilin Zhang f, Zhenzhen Wang a,b, Gongqi Yu a,b, Yongxiang Yu a,b, Peipei Qin a,b,e, Weizhi Wu c,d, Futang Pan c,d, Hong Liu a,b,c,d, Furen Zhang a,b,c,d,* a
Shandong Provincial Institute of Dermatology and Venereology, Shandong Academy of Medical Sciences, Jinan, Shandong, China Shandong Provincial Key Lab for Dermatovenereology, Jinan, Shandong, China c Shandong Provincial Hospital for Skin Diseases, Shandong University, Jinan, Shandong, China d Shandong Provincial Medical Center for Dermatovenereology, Jinan, Shandong, China e School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, Shandong, China f School of Medicine, Shandong University, Jinan, Shandong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 April 2014 Received in revised form 7 August 2014 Accepted 8 August 2014
Background: Generalized pustular psoriasis (GPP) is a rare type of psoriasis with potentially life-threatening implications. Mutations in IL36RN gene have been suggested to be causative or predisposing factors for GPP. Objective: To evaluate the genetic heterogeneity of GPP, PV and GPP alone, GPP with PV. Methods: We performed a sanger sequencing identify IL36RN mutations in 62 Chinese Han patients with sporadic GPP, including 17 GPP without psoriasis vulgaris (PV) (GPP alone) cases vs. 45 GPP with preceding, later or accompanied by PV (GPP with PV) cases; 16 patients with pediatric-onset GPP (PGPP) vs. 46 adult-onset GPP (AGPP). We included 96 healthy controls and 174 sporadic patients with PV. Resuts: We found 2 new variants and 4 known IL36RN variants in 29 GPP patients, 18 individuals carried recessive (homozygous/compound heterozygous) alleles and 11 cases harbored a single heterozygous change. Twelve PV patients and six controls harbored a single heterozygous for three out of the six variants. Significant differences were observed between GPP and PV groups, GPP alone and GPP with PV groups when compared frequencies of IL36RN variants, but we did not found association between PGPP and AGPP groups. Conclusion: Our study provided more evidence that GPP and PV are distinct subtypes of psoriasis caused by different pathogenesis, and GPP alone could be regarded as an especial entities of GPP which is different from GPP with PV on the etiology. ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Generalized pustular psoriasis (GPP) Psoriasis vulgaris (PV) GPP without psoriasis PV (GPP alone) GPP with preceding, later or accompanied by PV (GPP with PV) Interleukin 36 receptor antagonist (IL36RN) Mutation analysis
1. Introduction Generalized pustular psoriasis (GPP) is a rare type of psoriasis characterized by sudden and repeated episodes of generalized rash and disseminated pustules, often accompanied by high-grade fever. GPP could be divided into different subtypes: GPP without psoriasis vulgaris (PV) (GPP alone) vs. GPP with PV [1], and pediatric-onset GPP (PGPP) vs. adult-onset GPP (AGPP) [2]. The susceptibility background of the host has been proven to play a critical role in the onset and development of GPP. Marrakchi
* Corresponding author at: Shandong Provincial Institute of Dermatology and Venereology, 27397 Jingshi Road, Jinan, Shandong Province 250022, China. Tel.: +86 531 87298801; fax: +86 531 87984734. E-mail addresses: [email protected], [email protected] (F. Zhang).
et al. firstly reported a homozygous mutation p.Leu27Pro of IL36RN gene in familial GPP patients [3]. Since then the widely use of sequencing has led to identify 14 mutations of IL36RN as causative or predisposing factors in some sporadic cases of GPP [1,2,4–9]. And also diverse genetic heterogeneity has been reported in different subtypes of GPP: the associations of these mutations were stronger with the GPP alone form than with the GPP with PV form in Japanese population [1]; the percentage of IL36RN mutations of PGPP patients was much higher than that of AGPP patients in Chinese population [2]. On the other hand, although GPP is traditionally held to be a variant of psoriasis, it has been demonstrated that loss of IL36RN function did not confer susceptibility to PV in British and Chinese populations [2,7,10], implying that heritable factors might play different roles in GPP and PV patients.
http://dx.doi.org/10.1016/j.jdermsci.2014.08.007 0923-1811/ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
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Table 1 Descriptive characterization of patients with GPP, PV and control. Type
Male/female
Age (years) mean SD
Age at onset (years) of GPP mean SD
Number
GPP GPP alone GPP with PV PGPP AGPP PV Control
38/24 9/8 29/16 8/8 30/16 88/86 50/46
36.70 17.54 31.98 23.00 38.48 14.91 16.02 12.39 43.89 12.69 45.99 20.79 40.23 8.97
33.10 19.77 23.50 24.46 36.73 16.59 6.15 4.94 42.48 13.18 29.31 21.83a
62 17 45 16 46 174 96
GPP: generalized pustular psoriasis; PV: psoriasis vulgaris; GPP alone: GPP without PV; GPP with PV: GPP with preceding or accompanied by PV; PGPP: pediatric-onset generalized pustular psoriasis; AGPP: adult-onset generalized pustular psoriasis. a Age at onset (years) of PV mean SD.
Taken together, may GPP with PV be a severe form of PV with pustule outbreak? More evidence remains clarified in different populations. In order to evaluate the genetic heterogeneity of GPP, PV and GPP alone, GPP with PV, we have performed a sanger sequencing to identify IL36RN mutations in Chinese Han sporadic GPP and PV patients. 2. Materials and methods 2.1. Subjects A total of 62 sporadic and non-consanguineous GPP, 174 with PV and 96 controls from Chinese Han were included in the analysis. All GPP patients showed generalized erythema and disseminated pustules, often accompanied by high-grade fever, neutrophilia, and elevated C-reactive protein. Skin biopsy showed spongiform pustules of Kogoj in the subcorneal portion of the epidermis. GPP cases were classified into two sets of groups: GPP
without PV (GPP alone; 17 cases); GPP with preceding, later or accompanied by PV (GPP with PV; 45 cases); patients who developed GPP before the age of 18 years were further designated as pediatric-onset GPP (PGPP; 16 cases) vs. others were recognized as adult-onset GPP (AGPP; 46 cases). Individuals in the PV group had at least two psoriatic scales or a single scale occupying at least 1% of the total body surface outside the scalp, and all had a clinical history of psoriasis at least 10 years. All patients were diagnosed by experienced dermatologists based on typical clinical presentations or histopathological findings. Controls were unaffected individuals with no family history of psoriasis, recruited from the Health Examination Center. The characteristics of the different groups are summarized in Table 1, as well as described in Fig. 1. This study was approved by the human medical and ethics committee of Shandong Provincial Institute of Dermatology and Venereology. We have received signed forms of informed consent from all the participants in this study.
Fig. 1. Representative clinical and pathological features of psoriasis vulgaris and generalized pustular psoriasis. (a, b) Psoriasis vulgaris (PV). Well-demarcated scaly, red plaques are observed on the trunk. Cutaneous inflammation progresses to exfoliative dermatitis over the legs. (c, d) Generalized pustular psoriasis (GPP). Pustules on background erythema are seen on the trunk and arms. (e) Histopathological features of a typical psoriatic plaque in a PV patient. Characteristic features of psoriasis, acanthosis, hyperkeratosis and parakeratosis are seen in the epidermis. (f) Histopathological features of pustular lesions in patient with GPP. The epidermis shows thickening with regular elongation of rete ridges. The characteristic spongiform pustules of Kogoj are clearly observed in the epidermis of pustular lesions.
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Table 2 Primer sequences of PCR amplification for 4 coding exons of IL36RN. IL36RN Exon number
Forward primer
Reverse primer
Annealing temperature (8C)
PCR production size (bp)
2 3 4 5
50 -CTCAGCCTCTCTCTCCATGATT-30 50 -TGCTGTTACTTCTGGCACAGT-30 50 -CTGCTGAGAAGCCTCCCTTC-30 50 -GATTCTGTTGATGGCAGCTTT-30
50 -CGGGTCTATCCAGAACTCACTT-30 50 -CCAAGGAAAGTGGGTCATGT-30 50 -CTGAGTCCCCAGTGAGGATG-30 50 -CATGGGTCTCCTCCACTCAC-30
58 56.5 59.5 56.5
164 250 283 392
2.2. Mutation detection After informed consent, genomic DNA were extracted from all of the samples’ peripheral blood using QuickGene DNA whole blood kit L (Kurabo Industries Ltd., Japan). The four coding exons of IL36RN gene including its intron–exon boundaries were amplified by polymerase chain reaction (PCR) using primers. After amplification, products were purified and sequenced on an ABI 3130xl Genetic Analyser (Applied Biosystems ABI, Carlsbad, CA, USA). The primers of IL36RN (coding exons 2–5 of IL36RN according to RefSeq NM_012275.2) for PCR amplification were designed by Primer3 (http://primer3.ut.ee/) (Table 2) and synthesized by BGI (Shenzhen, China). The four fragments were amplified by PCR. The genotyping was done by using SNaPshot (ABI Prism SNaPshot multiplex kit; Applied Biosystems) on an ABI PRISM 3130xl genetic analyser and analyzed by ABI GeneScan software. 2.3. Statistical analysis The count numbering of the mutations detected in this study is on the basis of RefSeq NM_012275.2. Differences in frequencies of IL36RN mutations between groups were analyzed by 2 2
cross-table x2 test and Fisher’s exact test by using SPSS Statistics 17.0 software. P < 0.05 was recognized as significant threshold. 3. Results 3.1. Analysis of IL36RN mutations between GPP, PV patients and healthy controls Two new variants, c.169G>A (p.Val57Ile) and c.245C>T (p.Pro82Leu), and four known variants, c.115 + 6T>C (p.Arg10ArgX1), c.140A>G (p.Asn47Ser), c.227C>T (p.Pro76Leu) and c.368C>T (p.Thr123Met) were identified in 29 GPP patients (Fig. 2). Among of them, 18 individuals carried recessive (homozygous/compound heterozygous) alleles and 11 harbored a single heterozygous change (Table 3). Among of the six variants, c.115 + 6T>C and p.Asn47Ser at a single heterozygous change were also found in 12 PV patients. Three of them called c.115 + 6T>C, p.Asn47Ser and p.Val57Ile at heterozygous state were found in six healthy controls (Tables 4A and 4B). The total frequency of all IL36RN variants was 46.77% in GPP patients, 6.90% in PV patients and 6.25% in controls. A significant association between IL36RN variants and GPP patients could be observed when compared GPP group with PV and controls
Fig. 2. Sequencing data of IL36RN variants in patients with GPP and PV (GPP: generalized pustular psoriasis; PV: psoriasis vulgaris). (a) c.115 + 6T>C homozygote; (b) c.115 + 6T>C heterozygote; (c) c.140A>G homozygote; (d) c.140A>G heterozygote; (e) c.169G>A heterozygote; (f) c.227C>T heterozygote; (g) c.245C>T heterozygote; (h) c.368C>T heterozygote. Black arrow indicates the location of the variant.
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Table 3 Clinical data of GPP patients who carried IL36RN variants in our study. Gender
Age of onset of PV (years)
Age of onset of GPP (years)
Age (years)
Triggering factors
IL36RN variants
M F M F F F M M M F M
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
3 68 56 0.17 11 27 3 13 53 32 4.5
44 68 56 3.75 15 40 8 23 68 42 24
Tonsillitis Unknown No obvious triggering factors No obvious triggering factors Cold Pregnancy Upper respiratory tract infection No obvious triggering factors Improper use of hormone Cold/pregnancy Cold
GPP alone17 (PGPP)
F
N/A
0.3
17
No obvious triggering factors
GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP
M M M M F M F M M M F F F M M M M
N/A N/A N/A 71 1 50 26 32 12 16 25 33 17 26 25 31 20
3.75 11 56 78 21 60 46 52 25 39 48 38 19 27 43 38 40
Cold No obvious triggering factors Stress Unknown No obvious triggering factors Improper use of hormone Cold Cold No obvious triggering factors No obvious triggering factors Improper use of hormone No obvious triggering factors Damp No obvious triggering factors Tired No obvious triggering factors Cold
hom.c.115 + 6T>C het.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C + het.p.Pro76Leu het.c.115 + 6T>C + het.p.Thr123Met hom.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C het.p.Asn47Ser hom.c.115 + 6T>C het.p.Pro82Leu hom.p.Asn47Ser het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C het.p.Val57Ile hom.p.Asn47Ser het.c.115 + 6T>C
ID GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP
alone01 alone02 alone03 alone08 alone09 alone10 alone11 alone12 alone13 alone14 alone16
with with with with with with with with with with with with with with with with with
(PGPP) (AGPP) (AGPP) (PGPP) (PGPP) (AGPP) (PGPP) (PGPP) (AGPP) (AGPP) (PGPP)
PV01 PV02 PV03 PV06 PV07 PV11 PV13 PV16 PV18 PV22 PV25 PV26 PV29 PV30 PV35 PV37 PV41
(PGPP) (PGPP) (AGPP) (AGPP) (PGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP)
2 11 56 78 8 60 46 52 25 39 48 38 19 26 43 38 20
GPP alone: generalized pustular psoriasis without psoriasis vulgaris; GPP with PV: generalized pustular psoriasis with preceding or accompanied by psoriasis vulgaris; GPP: generalized pustular psoriasis; PV: psoriasis vulgaris. M = male; F = female; N/A = not associated; hom. = homozygous for, het. = heterozygous for.
(P = 1.11 1012, odds ratio (OR) = 11.86; P = 2.11 109, OR = 13.18, respectively) (Table 4A). Among of all the variants, only the most common one c.115 + 6T>C, whose frequency was 38.71% in GPP patients, 2.30% in PV patients and 1.04% in controls, showed significant association with GPP when compared with PV and controls (P = 2.68 1014, OR = 26.84; P = 2.38 1010, OR = 60.00, respectively) (Table 4A). Other five rarer variants showed no relationship when analyzed individually (all P > 0.05) (Table 4B). When compared PV to controls, no significant association was found (P = 0.84, OR = 1.11) (Table 4A).
3.2. Subgroup analysis of IL36RN mutations The subgroup analysis of GPP alone and GPP with PV revealed significant evidence of heterogeneity at all IL36RN variants and c.115 + 6T>C (70.59% vs. 37.78%, P = 0.02, OR = 3.95; 70.59% vs. 26.67%, P = 2.0 103, OR = 6.60, respectively) (Table 4A). We also compared the frequencies of variants in PGPP (pediatric-onset GPP) and AGPP (adult-onset GPP) patients, no significant association was found to associate with all IL36RN variants (P = 0.14, OR = 2.37). Only c.115 + 6T>C showed nominal evidence of association (P = 0.02, OR = 3.81) (Table 4A).
Table 4A The c.115 + 6T>C (p.Arg10ArgX1) variant and All IL36RN variants occurred in controls and in patients with GPP and PV. Genotype
c.115 + 6T>C (p.Arg10ArgX1) con
All IL36RN variants
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
PV
con
GPP GPP alone
GPP with PV
PGPP
AGPP
All GPP
PV
AA Aa aa Total
95 1 0 96
5 3 9 17
33 6 6 45
6 1 9 16
32 8 6 46
38 9 15 62
170 4 0 174
90 6 0 96
5 3 9 17
28 9 8 45
6 1 9 16
27 11 8 46
33 12 17 62
162 12 0 174
Variants frequency (%) P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
1.04
70.59
26.67
62.50
30.43
38.71
2.30
6.25
70.59
37.78
62.50
41.30
46.77
6.90
6.29E13
4.63E07
2.38E10
0.79
2.59E08
2.86E07
2.11E09
0.84
3.52E15 4.42E06 0.002
0.02
2.58E10 2.32E06 0.02
0.14 2.68E14
1.11E12
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Table 4B The other five rarer IL36RN variants who showed no relationship when analyzed individually both in patients with GPP and PV. Genotype
p.Asn47Ser (c.140A>G) con
p.Val57Ile (c.169G>A)
GPP
All GPP GPP with PV
PGPP
AGPP
con
p.Pro76Leu (c.227C>T)
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
PV
con
GPP GPP alone
GPP with PV
PGPP
AGPP
All GPP
PV
AA Aa aa Total
92 4 0 96
17 0 0 17
42 1 2 45
16 0 0 16
43 1 2 46
59 1 2 62
166 8 0 174
95 1 0 96
17 0 0 17
44 1 0 45
16 0 0 16
45 1 0 46
61 1 0 62
174 0 0 174
96 0 0 96
16 1 0 17
45 0 0 45
15 1 0 16
46 0 0 46
61 1 0 62
174 0 0 174
Variants frequency (%) P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
4.17
0
6.67
0
6.52
4.84
4.6
1.04
0
2.22
0
2.17
1.61
0
0
5.88
0
6.25
0
1.61
0
1
0.83 0.56
1
0.85
1
1
0.54
1
0.55
1
0.36
Genotype
P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
0.14
0.39
0.27 1
0.26
1
0.26
p.Pro82Leu (c.245C>T)
96 0 0 96 0
0.15
1 0.56
con
AA Aa aa Total Variants frequency (%)
1
0.26
p.Thr123Met (c.368C>T)
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
17 0 0 17 0
44 1 0 45 2.22
16 0 0 16 0
45 1 0 46 2.17
61 1 0 62 1.61
0.32
0.39
0.32
PV
174 0 0 174 0
con
96 0 0 96 0
GPP GPP alone
GPP with PV
PGPP
AGPP
16 1 0 17 5.88
45 0 0 45 0
15 1 0 16 6.25
46 0 0 46 0
0.15
1
0.14
All GPP
PV
61 1 0 62 1.61
174 0 0 174 0
0.39
0.27 1
0.26 0.26
0.26
Con: control; GPP alone: generalized pustular psoriasis without psoriasis vulgaris; GPP with PV: generalized pustular psoriasis with preceding or accompanied by psoriasis vulgaris; PGPP: pediatric-onset generalized pustular psoriasis; AGPP: adult-onset generalized pustular psoriasis; GPP: generalized pustular psoriasis; PV: psoriasis vulgaris. All the P value listed above was calculation based on 2 2 x2 test.
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GPP alone
PV
X. Li et al. / Journal of Dermatological Science 76 (2014) 132–138
3.3. The function prediction of the two new variants occurred in our study We found two new variants, p.Val57Ile and p.Pro82Leu, which have not been reported previously. P.Pro82Leu was only found in a single heterozygous state in GPP with PV11, whose effect was estimated to be damaging using Sorting Intolerant From Tolerant (SIFT) (http://sift.bii.a-star.edu.sg) pathogenicity prediction tool, neutral by Protein Variation Effect Analyzer (PROVEAN) (http:// provean.jcvi.org/index.php). The effects of p.Val57Ile were Neutral evaluated by PROVEAN and Tolerated by SIFT. 4. Discussion Up to date, 14 IL36RN mutations have been identified to date in GPP patients from African, European and Asian populations (p.Arg10X, p.Leu27Pro, p.His32Arg, p.Lys35Arg, c.115 + 6T>C, p.Asn47Ser, p.Arg48Trp, p.Pro76Leu, p.Glu94X, p.Arg102Gln, p.Arg102Trp, p.Ser113Leu, p.Thr123Arg and p.Thr123Met). In light of this, GPP in these populations has been shown to be related to DITRA caused by IL36RN mutations. The IL36RN gene encodes interleukin 36 receptor antagonist (IL36Ra), which belongs to the interleukin (IL)-1 family and has been shown to act as an antagonist of IL-36a, IL-36b, and IL-36g [11], thus avoiding exacerbated inflammatory responses [12,13]. Blumberg et al. found that the skin-specific overexpression of IL-36a in transgenic mice caused a psoriasis-like phenotype with pustular lesions which was exacerbated by a concomitant deficiency in IL36Ra. They also found that IL36R blockade resolved the inflammatory changes in human psoriatic skin transplanted into immunodeficient mice [14,15]. Anakinra, an IL-1 receptor antagonist, has been shown to be effective against GPP [16,17]. These findings support a role for IL36RN as a disease-causing gene in GPP. Here, we totally identified six variants and only two new variants. As the results of the function prediction, and both of the two variants were found in GPP with PV group, we could not include them as GPP causative variants, so we considered that the p.Val57Ile variant may be a SNP in the Chinese population. On the other hand, the p.Pro82Leu variant was not found in PV and control groups, together with the results of the pathogenicity prediction, this variant may lead to stimulate nuclear factor-kB (NF-kB) and mitogen-activated protein (MAP) kinases, and then exacerbated inflammatory responses via affecting the protein structure or the interaction with its receptor, but the exactly function remains unknown. The other variants, c.115 + 6T>C, p.Asn47Ser and p.Pro76Leu have been reported by Li et al., which showed similar frequencies with ours [2]. Interestingly, we found GPP alone16 was heterozygous for p.Pro76Leu plus homozygous for c.115 + 6T>C, similar to which has been reported previously only in Malay and Chinese population [2,8]. In addition, c.115 + 6T>C has also been reported only in Japanese and Malay populations but not in European and African populations, providing more evidence that both c.115 + 6T>C and c.227C>T–c.115 + 6T>C allele may be the primary variant in Asian populations. The c.115 + 6T>C, could disrupt the splicing of exon 3, leading to the synthesis of a truncated protein and harbored the development of GPP [4,8]. It has been demonstrated that GPP and PV are genetically distinct in various populations: some team like Onoufriadis’ and Berki’s from United Kingdom declared that in the British population loss of IL36RN activity was not associated with a significant increase of PV risk [7,10,18]; Li’s team reported that in Chinese population, there existed no significant association between IL36RN mutations and PV (P > 0.05). In the present study, the IL36RN mutations were unlikely to contribute to PV susceptibility in our individuals, agreed with the results mentioned above. Keratinocytes in PV patients have been shown to
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overexpress IL-36 [19,20], and given that our data indicated that the mechanisms of IL-36 overexpression differed between PV and GPP, we could not discount the possibility that mutations in functionally-related genes such as IL-36a, IL-36b, and IL-36g may be associated with PV. Sugiura et al. reported the different genetic factor between two subtypes of GPP for the first time in Japanese population: the majority of GPP alone (9 out of 11) cases have IL36RN mutations, but most cases of GPP with PV (17 out of 20) have no IL36RN mutation, and they put forward that GPP with PV might be a server syndrome of PV [1]. In our study, twelve out of 17 GPP alone and 17 out of 45 GPP with PV harbored IL36RN variants, a significant difference was also observed when compared the two subtypes of GPP, giving more evidences that IL36RN variants play different roles in the two subtypes of GPP patients on the etiology, implying the genetic heterogeneity between GPP alone and GPP with PV in Chinese population. It was notable that the frequency of c.115 + 6T>C differed significantly between PGPP and AGPP groups, but no significant association was found when referring to all IL36RN variants, different from the results of Li’s result [2]. We attributed the discrepancy to the imbalance sample sizes in our PGPP and AGPP patients (16 vs. 46). In conclusion, we identified two new variants and confirmed four known IL36RN variants in Chinese GPP patients and significant differences were observed between GPP and PV groups, GPP alone and GPP with PV groups, providing more evidence that GPP and PV are distinct subtypes of psoriasis caused by different pathogenesis, and GPP alone could be regarded as an especial entities of GPP which is different from GPP with PV on the etiology. Funding support This study was funded by a grant from the National Natural Science Foundation of China (81071288, 81072391, 81101187, 81271746, 31200933) and the Natural Science Foundation of Shandong Province (ZR2011HQ003, ZR2012HQ031). Acknowledgements We thank the individuals who participated in this project. This work was funded by a grant from the National Natural Science Foundation of China (81071288, 81072391, 81101187, 81271746, 31200933) and the Natural Science Foundation of Shandong Province (ZR2011HQ003, ZR2012HQ031). References [1] Sugiura 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;133:2514–21. [2] Li M, Han J, Lu Z, Li H, Zhu K, Cheng R, et al. Prevalent and rare mutations in IL36RN gene in Chinese patients with generalized pustular psoriasis and psoriasis vulgaris. J Invest Dermatol 2013;133:2637–9. [3] Marrakchi S, Guigue P, Renshaw BR, Puel A, Pei XY, Fraitag S, et al. Interleukin36-receptor antagonist deficiency and generalized pustular psoriasis. N Engl J Med 2011;365:620–8. [4] Farooq M, Nakai H, Fujimoto A, Fujikawa H, Matsuyama A, Kariya N, et al. Mutation analysis of the IL-36RN gene in 14 Japanese patients with generalized pustular psoriasis. Hum Mutat 2012;34:176–83. [5] Kanazawa N, Nakamura T, Mikita N, Furukawa F. Novel IL36RN mutation in a Japanese case of early onset generalized pustular psoriasis. J Dermatol 2013;40:749–51. [6] Ko¨rber A, Mo¨ssner R, Renner R, Sticht H, Wilsmann-Theis D, Schulz P, et al. Mutations in IL36RN in patients with generalized pustular psoriasis. J Invest Dermatol 2013;133:2634–7. [7] Onoufriadis A, Simpson MA, Pink AE, Di Meglio P, Smith CH, 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;89:432–7.
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[8] Setta-Kaffetzi N, Navarini AA, Patel VM, Pullabhatla V, Pink AE, Choon SE, et al. Rare pathogenic variants in IL36RN underlie a spectrum of psoriasis-associated pustular phenotypes. J Invest Dermatol 2013;133:1366–9. [9] Sugiura K, Takeichi T, Kono M, Ogawa Y, Shimoyama Y, Muro Y, et al. A novel IL-36RN/IL1F5 homozygous nonsense mutation, p.Arg10X, in a Japanese patient with adult-onset generalized pustular psoriasis. Br J Dermatol 2012;167:699–701. [10] Berki DM, Mahil SK, Burden AD, Trembath RC, Smith CH, Capon F, et al. Loss of IL36RN function does not confer susceptibility to psoriasis vulgaris. J Invest Dermatol 2014;134:271–3. [11] Towne JE, Garka KE, Renshaw BR, Virca GD, Sims JE. Interleukin (IL)-1F6 IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kappaB and MAPKs. J Biol Chem 2004;279:13677–88. [12] Dinarello C, Arend W, Sims J, Smith D, Blumberg H, O’Neill L, et al. IL-1 family nomenclature. Nat Immunol 2010;11:973. [13] Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010;10:89–102. [14] Blumberg H, Dinh H, Trueblood ES, Pretorius J, Kugler D, Weng N, et al. Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation. J Exp Med 2007;204:2603–14.
[15] Blumberg H, Dinh H, Dean Jr C, Trueblood ES, Bailey K, Shows D, et al. IL-1RL2 and its ligands contribute to the cytokine network in psoriasis. J Immunol 2010;185:4354–62. [16] Viguier M, Guigue P, Pages C, Smahi A, Bachelez H. Successful treatment of generalized pustular psoriasis with the interleukin-1-receptor antagonist anakinra: lack of correlation with IL1RN mutations. Ann Intern Med 2010;153:66–7. [17] Hu¨ffmeier U, Wa¨tzold M, Mohr J, Scho¨n MP, Mo¨ssner R. Successful therapy with anakinra in a patient with generalized pustular psoriasis carrying IL36RN mutations. Br J Dermatol 2014;170:202–4. [18] Strange A, Capon F, Spencer CC, Knight J, Weale ME, Allen MH, et al. A genomewide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 2010;42:985–90. [19] Johnston A, Xing X, Guzman AM, Riblett M, Loyd CM, Ward NL, et al. IL-1F5, -F6, F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression. J Immunol 2011;186:2613–22. [20] Carrier Y, Ma HL, Ramon HE, Napierata L, Small C, O’Toole M, et al. Interregulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis. J Invest Dermatol 2011;131:2428–37.
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Mutation analysis of the IL36RN gene in Chinese patients with generalized pustular psoriasis with/without psoriasis vulgaris Xiuyan Li a,b,e, Mingfei Chen a,b, Xi’an Fu a,b, Qilin Zhang f, Zhenzhen Wang a,b, Gongqi Yu a,b, Yongxiang Yu a,b, Peipei Qin a,b,e, Weizhi Wu c,d, Futang Pan c,d, Hong Liu a,b,c,d, Furen Zhang a,b,c,d,* a
Shandong Provincial Institute of Dermatology and Venereology, Shandong Academy of Medical Sciences, Jinan, Shandong, China Shandong Provincial Key Lab for Dermatovenereology, Jinan, Shandong, China c Shandong Provincial Hospital for Skin Diseases, Shandong University, Jinan, Shandong, China d Shandong Provincial Medical Center for Dermatovenereology, Jinan, Shandong, China e School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, Shandong, China f School of Medicine, Shandong University, Jinan, Shandong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 April 2014 Received in revised form 7 August 2014 Accepted 8 August 2014
Background: Generalized pustular psoriasis (GPP) is a rare type of psoriasis with potentially life-threatening implications. Mutations in IL36RN gene have been suggested to be causative or predisposing factors for GPP. Objective: To evaluate the genetic heterogeneity of GPP, PV and GPP alone, GPP with PV. Methods: We performed a sanger sequencing identify IL36RN mutations in 62 Chinese Han patients with sporadic GPP, including 17 GPP without psoriasis vulgaris (PV) (GPP alone) cases vs. 45 GPP with preceding, later or accompanied by PV (GPP with PV) cases; 16 patients with pediatric-onset GPP (PGPP) vs. 46 adult-onset GPP (AGPP). We included 96 healthy controls and 174 sporadic patients with PV. Resuts: We found 2 new variants and 4 known IL36RN variants in 29 GPP patients, 18 individuals carried recessive (homozygous/compound heterozygous) alleles and 11 cases harbored a single heterozygous change. Twelve PV patients and six controls harbored a single heterozygous for three out of the six variants. Significant differences were observed between GPP and PV groups, GPP alone and GPP with PV groups when compared frequencies of IL36RN variants, but we did not found association between PGPP and AGPP groups. Conclusion: Our study provided more evidence that GPP and PV are distinct subtypes of psoriasis caused by different pathogenesis, and GPP alone could be regarded as an especial entities of GPP which is different from GPP with PV on the etiology. ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Generalized pustular psoriasis (GPP) Psoriasis vulgaris (PV) GPP without psoriasis PV (GPP alone) GPP with preceding, later or accompanied by PV (GPP with PV) Interleukin 36 receptor antagonist (IL36RN) Mutation analysis
1. Introduction Generalized pustular psoriasis (GPP) is a rare type of psoriasis characterized by sudden and repeated episodes of generalized rash and disseminated pustules, often accompanied by high-grade fever. GPP could be divided into different subtypes: GPP without psoriasis vulgaris (PV) (GPP alone) vs. GPP with PV [1], and pediatric-onset GPP (PGPP) vs. adult-onset GPP (AGPP) [2]. The susceptibility background of the host has been proven to play a critical role in the onset and development of GPP. Marrakchi
* Corresponding author at: Shandong Provincial Institute of Dermatology and Venereology, 27397 Jingshi Road, Jinan, Shandong Province 250022, China. Tel.: +86 531 87298801; fax: +86 531 87984734. E-mail addresses: [email protected], [email protected] (F. Zhang).
et al. firstly reported a homozygous mutation p.Leu27Pro of IL36RN gene in familial GPP patients [3]. Since then the widely use of sequencing has led to identify 14 mutations of IL36RN as causative or predisposing factors in some sporadic cases of GPP [1,2,4–9]. And also diverse genetic heterogeneity has been reported in different subtypes of GPP: the associations of these mutations were stronger with the GPP alone form than with the GPP with PV form in Japanese population [1]; the percentage of IL36RN mutations of PGPP patients was much higher than that of AGPP patients in Chinese population [2]. On the other hand, although GPP is traditionally held to be a variant of psoriasis, it has been demonstrated that loss of IL36RN function did not confer susceptibility to PV in British and Chinese populations [2,7,10], implying that heritable factors might play different roles in GPP and PV patients.
http://dx.doi.org/10.1016/j.jdermsci.2014.08.007 0923-1811/ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
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Table 1 Descriptive characterization of patients with GPP, PV and control. Type
Male/female
Age (years) mean SD
Age at onset (years) of GPP mean SD
Number
GPP GPP alone GPP with PV PGPP AGPP PV Control
38/24 9/8 29/16 8/8 30/16 88/86 50/46
36.70 17.54 31.98 23.00 38.48 14.91 16.02 12.39 43.89 12.69 45.99 20.79 40.23 8.97
33.10 19.77 23.50 24.46 36.73 16.59 6.15 4.94 42.48 13.18 29.31 21.83a
62 17 45 16 46 174 96
GPP: generalized pustular psoriasis; PV: psoriasis vulgaris; GPP alone: GPP without PV; GPP with PV: GPP with preceding or accompanied by PV; PGPP: pediatric-onset generalized pustular psoriasis; AGPP: adult-onset generalized pustular psoriasis. a Age at onset (years) of PV mean SD.
Taken together, may GPP with PV be a severe form of PV with pustule outbreak? More evidence remains clarified in different populations. In order to evaluate the genetic heterogeneity of GPP, PV and GPP alone, GPP with PV, we have performed a sanger sequencing to identify IL36RN mutations in Chinese Han sporadic GPP and PV patients. 2. Materials and methods 2.1. Subjects A total of 62 sporadic and non-consanguineous GPP, 174 with PV and 96 controls from Chinese Han were included in the analysis. All GPP patients showed generalized erythema and disseminated pustules, often accompanied by high-grade fever, neutrophilia, and elevated C-reactive protein. Skin biopsy showed spongiform pustules of Kogoj in the subcorneal portion of the epidermis. GPP cases were classified into two sets of groups: GPP
without PV (GPP alone; 17 cases); GPP with preceding, later or accompanied by PV (GPP with PV; 45 cases); patients who developed GPP before the age of 18 years were further designated as pediatric-onset GPP (PGPP; 16 cases) vs. others were recognized as adult-onset GPP (AGPP; 46 cases). Individuals in the PV group had at least two psoriatic scales or a single scale occupying at least 1% of the total body surface outside the scalp, and all had a clinical history of psoriasis at least 10 years. All patients were diagnosed by experienced dermatologists based on typical clinical presentations or histopathological findings. Controls were unaffected individuals with no family history of psoriasis, recruited from the Health Examination Center. The characteristics of the different groups are summarized in Table 1, as well as described in Fig. 1. This study was approved by the human medical and ethics committee of Shandong Provincial Institute of Dermatology and Venereology. We have received signed forms of informed consent from all the participants in this study.
Fig. 1. Representative clinical and pathological features of psoriasis vulgaris and generalized pustular psoriasis. (a, b) Psoriasis vulgaris (PV). Well-demarcated scaly, red plaques are observed on the trunk. Cutaneous inflammation progresses to exfoliative dermatitis over the legs. (c, d) Generalized pustular psoriasis (GPP). Pustules on background erythema are seen on the trunk and arms. (e) Histopathological features of a typical psoriatic plaque in a PV patient. Characteristic features of psoriasis, acanthosis, hyperkeratosis and parakeratosis are seen in the epidermis. (f) Histopathological features of pustular lesions in patient with GPP. The epidermis shows thickening with regular elongation of rete ridges. The characteristic spongiform pustules of Kogoj are clearly observed in the epidermis of pustular lesions.
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Table 2 Primer sequences of PCR amplification for 4 coding exons of IL36RN. IL36RN Exon number
Forward primer
Reverse primer
Annealing temperature (8C)
PCR production size (bp)
2 3 4 5
50 -CTCAGCCTCTCTCTCCATGATT-30 50 -TGCTGTTACTTCTGGCACAGT-30 50 -CTGCTGAGAAGCCTCCCTTC-30 50 -GATTCTGTTGATGGCAGCTTT-30
50 -CGGGTCTATCCAGAACTCACTT-30 50 -CCAAGGAAAGTGGGTCATGT-30 50 -CTGAGTCCCCAGTGAGGATG-30 50 -CATGGGTCTCCTCCACTCAC-30
58 56.5 59.5 56.5
164 250 283 392
2.2. Mutation detection After informed consent, genomic DNA were extracted from all of the samples’ peripheral blood using QuickGene DNA whole blood kit L (Kurabo Industries Ltd., Japan). The four coding exons of IL36RN gene including its intron–exon boundaries were amplified by polymerase chain reaction (PCR) using primers. After amplification, products were purified and sequenced on an ABI 3130xl Genetic Analyser (Applied Biosystems ABI, Carlsbad, CA, USA). The primers of IL36RN (coding exons 2–5 of IL36RN according to RefSeq NM_012275.2) for PCR amplification were designed by Primer3 (http://primer3.ut.ee/) (Table 2) and synthesized by BGI (Shenzhen, China). The four fragments were amplified by PCR. The genotyping was done by using SNaPshot (ABI Prism SNaPshot multiplex kit; Applied Biosystems) on an ABI PRISM 3130xl genetic analyser and analyzed by ABI GeneScan software. 2.3. Statistical analysis The count numbering of the mutations detected in this study is on the basis of RefSeq NM_012275.2. Differences in frequencies of IL36RN mutations between groups were analyzed by 2 2
cross-table x2 test and Fisher’s exact test by using SPSS Statistics 17.0 software. P < 0.05 was recognized as significant threshold. 3. Results 3.1. Analysis of IL36RN mutations between GPP, PV patients and healthy controls Two new variants, c.169G>A (p.Val57Ile) and c.245C>T (p.Pro82Leu), and four known variants, c.115 + 6T>C (p.Arg10ArgX1), c.140A>G (p.Asn47Ser), c.227C>T (p.Pro76Leu) and c.368C>T (p.Thr123Met) were identified in 29 GPP patients (Fig. 2). Among of them, 18 individuals carried recessive (homozygous/compound heterozygous) alleles and 11 harbored a single heterozygous change (Table 3). Among of the six variants, c.115 + 6T>C and p.Asn47Ser at a single heterozygous change were also found in 12 PV patients. Three of them called c.115 + 6T>C, p.Asn47Ser and p.Val57Ile at heterozygous state were found in six healthy controls (Tables 4A and 4B). The total frequency of all IL36RN variants was 46.77% in GPP patients, 6.90% in PV patients and 6.25% in controls. A significant association between IL36RN variants and GPP patients could be observed when compared GPP group with PV and controls
Fig. 2. Sequencing data of IL36RN variants in patients with GPP and PV (GPP: generalized pustular psoriasis; PV: psoriasis vulgaris). (a) c.115 + 6T>C homozygote; (b) c.115 + 6T>C heterozygote; (c) c.140A>G homozygote; (d) c.140A>G heterozygote; (e) c.169G>A heterozygote; (f) c.227C>T heterozygote; (g) c.245C>T heterozygote; (h) c.368C>T heterozygote. Black arrow indicates the location of the variant.
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Table 3 Clinical data of GPP patients who carried IL36RN variants in our study. Gender
Age of onset of PV (years)
Age of onset of GPP (years)
Age (years)
Triggering factors
IL36RN variants
M F M F F F M M M F M
N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
3 68 56 0.17 11 27 3 13 53 32 4.5
44 68 56 3.75 15 40 8 23 68 42 24
Tonsillitis Unknown No obvious triggering factors No obvious triggering factors Cold Pregnancy Upper respiratory tract infection No obvious triggering factors Improper use of hormone Cold/pregnancy Cold
GPP alone17 (PGPP)
F
N/A
0.3
17
No obvious triggering factors
GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP
M M M M F M F M M M F F F M M M M
N/A N/A N/A 71 1 50 26 32 12 16 25 33 17 26 25 31 20
3.75 11 56 78 21 60 46 52 25 39 48 38 19 27 43 38 40
Cold No obvious triggering factors Stress Unknown No obvious triggering factors Improper use of hormone Cold Cold No obvious triggering factors No obvious triggering factors Improper use of hormone No obvious triggering factors Damp No obvious triggering factors Tired No obvious triggering factors Cold
hom.c.115 + 6T>C het.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C hom.c.115 + 6T>C + het.p.Pro76Leu het.c.115 + 6T>C + het.p.Thr123Met hom.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C het.p.Asn47Ser hom.c.115 + 6T>C het.p.Pro82Leu hom.p.Asn47Ser het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C hom.c.115 + 6T>C het.c.115 + 6T>C het.p.Val57Ile hom.p.Asn47Ser het.c.115 + 6T>C
ID GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP GPP
alone01 alone02 alone03 alone08 alone09 alone10 alone11 alone12 alone13 alone14 alone16
with with with with with with with with with with with with with with with with with
(PGPP) (AGPP) (AGPP) (PGPP) (PGPP) (AGPP) (PGPP) (PGPP) (AGPP) (AGPP) (PGPP)
PV01 PV02 PV03 PV06 PV07 PV11 PV13 PV16 PV18 PV22 PV25 PV26 PV29 PV30 PV35 PV37 PV41
(PGPP) (PGPP) (AGPP) (AGPP) (PGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP) (AGPP)
2 11 56 78 8 60 46 52 25 39 48 38 19 26 43 38 20
GPP alone: generalized pustular psoriasis without psoriasis vulgaris; GPP with PV: generalized pustular psoriasis with preceding or accompanied by psoriasis vulgaris; GPP: generalized pustular psoriasis; PV: psoriasis vulgaris. M = male; F = female; N/A = not associated; hom. = homozygous for, het. = heterozygous for.
(P = 1.11 1012, odds ratio (OR) = 11.86; P = 2.11 109, OR = 13.18, respectively) (Table 4A). Among of all the variants, only the most common one c.115 + 6T>C, whose frequency was 38.71% in GPP patients, 2.30% in PV patients and 1.04% in controls, showed significant association with GPP when compared with PV and controls (P = 2.68 1014, OR = 26.84; P = 2.38 1010, OR = 60.00, respectively) (Table 4A). Other five rarer variants showed no relationship when analyzed individually (all P > 0.05) (Table 4B). When compared PV to controls, no significant association was found (P = 0.84, OR = 1.11) (Table 4A).
3.2. Subgroup analysis of IL36RN mutations The subgroup analysis of GPP alone and GPP with PV revealed significant evidence of heterogeneity at all IL36RN variants and c.115 + 6T>C (70.59% vs. 37.78%, P = 0.02, OR = 3.95; 70.59% vs. 26.67%, P = 2.0 103, OR = 6.60, respectively) (Table 4A). We also compared the frequencies of variants in PGPP (pediatric-onset GPP) and AGPP (adult-onset GPP) patients, no significant association was found to associate with all IL36RN variants (P = 0.14, OR = 2.37). Only c.115 + 6T>C showed nominal evidence of association (P = 0.02, OR = 3.81) (Table 4A).
Table 4A The c.115 + 6T>C (p.Arg10ArgX1) variant and All IL36RN variants occurred in controls and in patients with GPP and PV. Genotype
c.115 + 6T>C (p.Arg10ArgX1) con
All IL36RN variants
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
PV
con
GPP GPP alone
GPP with PV
PGPP
AGPP
All GPP
PV
AA Aa aa Total
95 1 0 96
5 3 9 17
33 6 6 45
6 1 9 16
32 8 6 46
38 9 15 62
170 4 0 174
90 6 0 96
5 3 9 17
28 9 8 45
6 1 9 16
27 11 8 46
33 12 17 62
162 12 0 174
Variants frequency (%) P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
1.04
70.59
26.67
62.50
30.43
38.71
2.30
6.25
70.59
37.78
62.50
41.30
46.77
6.90
6.29E13
4.63E07
2.38E10
0.79
2.59E08
2.86E07
2.11E09
0.84
3.52E15 4.42E06 0.002
0.02
2.58E10 2.32E06 0.02
0.14 2.68E14
1.11E12
136
Table 4B The other five rarer IL36RN variants who showed no relationship when analyzed individually both in patients with GPP and PV. Genotype
p.Asn47Ser (c.140A>G) con
p.Val57Ile (c.169G>A)
GPP
All GPP GPP with PV
PGPP
AGPP
con
p.Pro76Leu (c.227C>T)
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
PV
con
GPP GPP alone
GPP with PV
PGPP
AGPP
All GPP
PV
AA Aa aa Total
92 4 0 96
17 0 0 17
42 1 2 45
16 0 0 16
43 1 2 46
59 1 2 62
166 8 0 174
95 1 0 96
17 0 0 17
44 1 0 45
16 0 0 16
45 1 0 46
61 1 0 62
174 0 0 174
96 0 0 96
16 1 0 17
45 0 0 45
15 1 0 16
46 0 0 46
61 1 0 62
174 0 0 174
Variants frequency (%) P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
4.17
0
6.67
0
6.52
4.84
4.6
1.04
0
2.22
0
2.17
1.61
0
0
5.88
0
6.25
0
1.61
0
1
0.83 0.56
1
0.85
1
1
0.54
1
0.55
1
0.36
Genotype
P P (GPP alone vs. GPP with PV) P (PGPP vs. AGPP) P (GPP vs. PV)
0.14
0.39
0.27 1
0.26
1
0.26
p.Pro82Leu (c.245C>T)
96 0 0 96 0
0.15
1 0.56
con
AA Aa aa Total Variants frequency (%)
1
0.26
p.Thr123Met (c.368C>T)
GPP
All GPP
GPP alone
GPP with PV
PGPP
AGPP
17 0 0 17 0
44 1 0 45 2.22
16 0 0 16 0
45 1 0 46 2.17
61 1 0 62 1.61
0.32
0.39
0.32
PV
174 0 0 174 0
con
96 0 0 96 0
GPP GPP alone
GPP with PV
PGPP
AGPP
16 1 0 17 5.88
45 0 0 45 0
15 1 0 16 6.25
46 0 0 46 0
0.15
1
0.14
All GPP
PV
61 1 0 62 1.61
174 0 0 174 0
0.39
0.27 1
0.26 0.26
0.26
Con: control; GPP alone: generalized pustular psoriasis without psoriasis vulgaris; GPP with PV: generalized pustular psoriasis with preceding or accompanied by psoriasis vulgaris; PGPP: pediatric-onset generalized pustular psoriasis; AGPP: adult-onset generalized pustular psoriasis; GPP: generalized pustular psoriasis; PV: psoriasis vulgaris. All the P value listed above was calculation based on 2 2 x2 test.
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GPP alone
PV
X. Li et al. / Journal of Dermatological Science 76 (2014) 132–138
3.3. The function prediction of the two new variants occurred in our study We found two new variants, p.Val57Ile and p.Pro82Leu, which have not been reported previously. P.Pro82Leu was only found in a single heterozygous state in GPP with PV11, whose effect was estimated to be damaging using Sorting Intolerant From Tolerant (SIFT) (http://sift.bii.a-star.edu.sg) pathogenicity prediction tool, neutral by Protein Variation Effect Analyzer (PROVEAN) (http:// provean.jcvi.org/index.php). The effects of p.Val57Ile were Neutral evaluated by PROVEAN and Tolerated by SIFT. 4. Discussion Up to date, 14 IL36RN mutations have been identified to date in GPP patients from African, European and Asian populations (p.Arg10X, p.Leu27Pro, p.His32Arg, p.Lys35Arg, c.115 + 6T>C, p.Asn47Ser, p.Arg48Trp, p.Pro76Leu, p.Glu94X, p.Arg102Gln, p.Arg102Trp, p.Ser113Leu, p.Thr123Arg and p.Thr123Met). In light of this, GPP in these populations has been shown to be related to DITRA caused by IL36RN mutations. The IL36RN gene encodes interleukin 36 receptor antagonist (IL36Ra), which belongs to the interleukin (IL)-1 family and has been shown to act as an antagonist of IL-36a, IL-36b, and IL-36g [11], thus avoiding exacerbated inflammatory responses [12,13]. Blumberg et al. found that the skin-specific overexpression of IL-36a in transgenic mice caused a psoriasis-like phenotype with pustular lesions which was exacerbated by a concomitant deficiency in IL36Ra. They also found that IL36R blockade resolved the inflammatory changes in human psoriatic skin transplanted into immunodeficient mice [14,15]. Anakinra, an IL-1 receptor antagonist, has been shown to be effective against GPP [16,17]. These findings support a role for IL36RN as a disease-causing gene in GPP. Here, we totally identified six variants and only two new variants. As the results of the function prediction, and both of the two variants were found in GPP with PV group, we could not include them as GPP causative variants, so we considered that the p.Val57Ile variant may be a SNP in the Chinese population. On the other hand, the p.Pro82Leu variant was not found in PV and control groups, together with the results of the pathogenicity prediction, this variant may lead to stimulate nuclear factor-kB (NF-kB) and mitogen-activated protein (MAP) kinases, and then exacerbated inflammatory responses via affecting the protein structure or the interaction with its receptor, but the exactly function remains unknown. The other variants, c.115 + 6T>C, p.Asn47Ser and p.Pro76Leu have been reported by Li et al., which showed similar frequencies with ours [2]. Interestingly, we found GPP alone16 was heterozygous for p.Pro76Leu plus homozygous for c.115 + 6T>C, similar to which has been reported previously only in Malay and Chinese population [2,8]. In addition, c.115 + 6T>C has also been reported only in Japanese and Malay populations but not in European and African populations, providing more evidence that both c.115 + 6T>C and c.227C>T–c.115 + 6T>C allele may be the primary variant in Asian populations. The c.115 + 6T>C, could disrupt the splicing of exon 3, leading to the synthesis of a truncated protein and harbored the development of GPP [4,8]. It has been demonstrated that GPP and PV are genetically distinct in various populations: some team like Onoufriadis’ and Berki’s from United Kingdom declared that in the British population loss of IL36RN activity was not associated with a significant increase of PV risk [7,10,18]; Li’s team reported that in Chinese population, there existed no significant association between IL36RN mutations and PV (P > 0.05). In the present study, the IL36RN mutations were unlikely to contribute to PV susceptibility in our individuals, agreed with the results mentioned above. Keratinocytes in PV patients have been shown to
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overexpress IL-36 [19,20], and given that our data indicated that the mechanisms of IL-36 overexpression differed between PV and GPP, we could not discount the possibility that mutations in functionally-related genes such as IL-36a, IL-36b, and IL-36g may be associated with PV. Sugiura et al. reported the different genetic factor between two subtypes of GPP for the first time in Japanese population: the majority of GPP alone (9 out of 11) cases have IL36RN mutations, but most cases of GPP with PV (17 out of 20) have no IL36RN mutation, and they put forward that GPP with PV might be a server syndrome of PV [1]. In our study, twelve out of 17 GPP alone and 17 out of 45 GPP with PV harbored IL36RN variants, a significant difference was also observed when compared the two subtypes of GPP, giving more evidences that IL36RN variants play different roles in the two subtypes of GPP patients on the etiology, implying the genetic heterogeneity between GPP alone and GPP with PV in Chinese population. It was notable that the frequency of c.115 + 6T>C differed significantly between PGPP and AGPP groups, but no significant association was found when referring to all IL36RN variants, different from the results of Li’s result [2]. We attributed the discrepancy to the imbalance sample sizes in our PGPP and AGPP patients (16 vs. 46). In conclusion, we identified two new variants and confirmed four known IL36RN variants in Chinese GPP patients and significant differences were observed between GPP and PV groups, GPP alone and GPP with PV groups, providing more evidence that GPP and PV are distinct subtypes of psoriasis caused by different pathogenesis, and GPP alone could be regarded as an especial entities of GPP which is different from GPP with PV on the etiology. Funding support This study was funded by a grant from the National Natural Science Foundation of China (81071288, 81072391, 81101187, 81271746, 31200933) and the Natural Science Foundation of Shandong Province (ZR2011HQ003, ZR2012HQ031). Acknowledgements We thank the individuals who participated in this project. This work was funded by a grant from the National Natural Science Foundation of China (81071288, 81072391, 81101187, 81271746, 31200933) and the Natural Science Foundation of Shandong Province (ZR2011HQ003, ZR2012HQ031). References [1] Sugiura 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;133:2514–21. [2] Li M, Han J, Lu Z, Li H, Zhu K, Cheng R, et al. Prevalent and rare mutations in IL36RN gene in Chinese patients with generalized pustular psoriasis and psoriasis vulgaris. J Invest Dermatol 2013;133:2637–9. [3] Marrakchi S, Guigue P, Renshaw BR, Puel A, Pei XY, Fraitag S, et al. Interleukin36-receptor antagonist deficiency and generalized pustular psoriasis. N Engl J Med 2011;365:620–8. [4] Farooq M, Nakai H, Fujimoto A, Fujikawa H, Matsuyama A, Kariya N, et al. Mutation analysis of the IL-36RN gene in 14 Japanese patients with generalized pustular psoriasis. Hum Mutat 2012;34:176–83. [5] Kanazawa N, Nakamura T, Mikita N, Furukawa F. Novel IL36RN mutation in a Japanese case of early onset generalized pustular psoriasis. J Dermatol 2013;40:749–51. [6] Ko¨rber A, Mo¨ssner R, Renner R, Sticht H, Wilsmann-Theis D, Schulz P, et al. Mutations in IL36RN in patients with generalized pustular psoriasis. J Invest Dermatol 2013;133:2634–7. [7] Onoufriadis A, Simpson MA, Pink AE, Di Meglio P, Smith CH, 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;89:432–7.
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