Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271 [2] Tarutani M, Itami S, Okabe M, Ikawa M, Tezuka T, Yoshikawa K, et al. Tissuespecific knockout of the mouse Pig-a gene reveals important roles for GPIanchored proteins in skin development. Proc Natl Acad Sci U S A 1997;94:7400– 5. [3] Vasioukhin V, Degenstein L, Wise B, Fuchs E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc Natl Acad Sci U S A 1999;96:8551–6. [4] Sano S, Itami S, Takeda K, Tarutani M, Yamaguchi Y, Miura H, et al. Keratinocytespecific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis. EMBO J 1999;18:4657–68. [5] Tarutani M, Nakajima K, Takaishi M, Ohko K, Sano S. Epidermal hyperplasia induced by Raf-MAPK signaling requires Stat3 activation. J Dermatol Sci 2013;72:110–5. [6] Ramirez A, Page A, Gandarillas A, Zanet J, Pibre S, Vidal M, et al. A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination. Genesis 2004;39:52–7. [7] Liu F, Woitge HW, Braut A, Kronenberg MS, Lichtler AC, Mina M, et al. Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues. Int J Dev Biol 2004;48:645–54. [8] Hafner M, Wenk J, Nenci A, Pasparakis M, Scharffetter-Kochanek K, Smyth N, et al. Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyteexpressed protein. Genesis 2004;38:176–81.

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Yujin Nakagawaa, Gyohei Egawaa,*, Yoshiki Miyachia, Kenji Kabashimaa,b a Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan; bPRESTO, Japan Science and Technology Agency, 7 Gobancho, Chiyoda-ku, Tokyo 102-0075, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara, Kyoto 606-8507, Japan. Tel.: +81 75 751 3605; fax: +81 75 761 3002 E-mail addresses: [email protected] (G. Egawa), [email protected] (K. Kabashima). Received 11 June 2014

http://dx.doi.org/10.1016/j.jdermsci.2014.10.006

Letter to the Editor Novel IL36RN gene mutation revealed by analysis of 8 Japanese patients with generalized pustular psoriasis To the Editor, Generalized pustular psoriasis (GPP) is the most aggressive form of psoriasis, and is characterized by recurrent febrile attacks with disseminated subcorneal pustules on generalized skin rashes [1]. GPP can develop in patients with psoriasis vulgaris (PV), palmoplantar pustulosis (PPP), or acrodermatitis continua of Hallopeua (ACH). In 2011, Marrakchi et al. identified a homozygous p.L27P mutation of IL36RN encoding an interleukin-36receptor antagonist (IL-36Ra) in Tunisian cases with familial GPP [2]. Onoufriadis et al. reported homozygous p.S113L and compound heterozygous p.S113L/p.R48T mutations of IL36RN in sporadic European GPP cases [3]. In 2012, Sugiura et al. reported for the first time a nonsense mutation (p.R10X) of IL36RN in an Asian population [4]. Farooq et al. performed mutation analysis of IL36RN in 14 Japanese patients with GPP, and identified mutations in two patients: compound heterozygous c.115 + 6T > C (p.R10RfsX1)/p.T123R and p.R10X/c.115 + 6T > C mutations [5]. In 2013, Sugiura et al. searched for IL36RN mutations in two groups of Japanese GPP patients, those having GPP without PV (11 cases) and GPP with PV (20 cases). Nine of the 11 GPP patients without PV had homozygous p.R10X or compound heterozygous p.R10X/c.115 + 6T > C mutations [6]. Recently, Kanazawa et al. reported a novel IL36RN mutation (compound heterozygous p.R10X/p.T123M) in a Japanese case of early onset GPP [1]. To gain a better understanding of the genetic components of GPP in the Japanese population, we carried out sequencing analysis of the IL36RN gene. We recruited a total of 8 patients with GPP (median age 38, 28–75 years, male:female ratio = 1.0:3.0) diagnosed according to Japanese Ministry of Health, Labor and Welfare criteria for GPP [7]. The basic characteristics of the GPP cases are shown in Table 1. The age of onset was younger than 10 years in two cases (Nos. 3 and 6). No cases had a familial history of GPP, and four cases (Nos. 1, 3, 4 and 7) had arthritis. PV (Nos. 2 and 4), PPP (No. 1) and ACH (No. 8) preceded GPP, and no skin lesions preceded GPP in four cases (Nos. 3, 5, 6 and 7). We further recruited a total of 258 patients with PV diagnosed by clinical and histopathological findings (median age 53, 11–85 years, male:female ratio = 2.4:1.0).

As controls, 1130 individuals who had never been diagnosed as PV or GPP were recruited from Fukui University and the Miyatake clinic (median age 46, 19–75 years, male:female ratio = 1.3:1.0). All individuals were unrelated Japanese and gave written informed consent to participate in the study. The study was approved by the ethical committees of the Institute of Physical and Chemical Research (RIKEN), the University of Tokyo, The Jikei University School of Medicine, Fukui University and the Miyatake clinic. Genomic DNA was prepared in accordance with standard protocols, and we carried out sequencing analysis of all five exons including exon–intron boundaries of the IL36RN gene in eight patients with GPP and 258 patients with PV using an ABI 3730xl DNA analyzer (Applied Biosystems) [5]. We found a total of 6 single nucleotide variants (SNVs), including 5 nonsynonymous SNVs and a SNV in intron 3 (Table 1, Supplementary Table 1 and Fig. 1). To access their allele frequencies in the general Japanese population, we genotyped the 6 SNVs in 1130 controls by multiplex-PCR-based Invader assay (Hologic). Supplementary Table 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.10.008. Recent studies have reported several IL36RN mutations in GPP patients, including p.R10X, p.L27P, p.H32R, p.L35R, c.115 + 6T > C, p.N47S, p.R48 W, p.P76L, p.E94X, p.R102Q, p.102Y, p.S113L, p.T123R and p.T123M [1,8,9], and four IL36RN mutations p.R10X, c.115 + 6T > C: p.R10RfsX1, p.T123R and p.T123M, have been reported in a Japanese population [1,5,6]. In this study, two SNVs, p.N47S and p.P82L, were observed in both PV cases and controls with allele frequencies > 1% (Supplementary Table 1); however, no statistically significant differences were observed in these two genotypes between PV cases and controls (data not shown). Both p.N47S and p.P82L were predicted to be deleterious by SIFT (http:// sift.jcvi.org/) but neutral by PROVEAN (http://sift.jcvi.org/). One control subject was homozygous for p.N47S and the possibility that this subject might develop GPP throughout life could not be completely excluded. c.334G > A (rs143724424) was not observed in the patients with PV or controls. In this study, we identified a novel IL36RN missense mutation, c.334G > A in exon 5, that caused p.E112K substitution and was located adjacent to a 113 amino acid residue. The missense c.338C > T mutation causing p.S113L substitution is the most prevalent allele in European GPP patients, and this variant is associated with an elevated proinflammatory response following

Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271

268

Table 1 Summary of the GPP patients analyzed in this study. Patient

Age

Sex

Age of onset

Family history

Arthritis

Preceding skin lesion

IL36RN mutation

1 2 3 4 5 6 7 8

51 28 38 44 75 38 64 63

F F F F M F M F

51 24 5 34 42 9 59 45

( ( ( ( ( ( ( (

(+) ( ) (+) (+) ( ) ( ) (+) ( )

PPP PV ( ) PV ( ) ( ) ( ) ACH

( ) ( ) ( ) ( ) ( ) ( ) c.28C > T hetero, c.334G > A hetero c.28C > T homo

) ) ) ) ) ) ) )

GPP: generalized pustular psoriasis, F: female, M: male, PPP: palmoplantar pustulosis, PV: psoriasis vulgaris, ACH: acrodermatitis continua of Hallopeau, hetero: heterozygous, homo: homozygous.

ex vivo stimulation with IL-36A, suggesting loss of function of IL36RN [3,10]. p.E112K was predicted to be deleterious and damaging by SIFT and PROVEAN, respectively. These findings suggested that the p.E112K mutation might influence IL36RN protein function. In case No. 7, we confirmed that the patient had c.28C > T:p.R10X mutation in one allele and c.334G > A:p.E112K mutation in another allele by cloning and sequencing of the IL36RN region. Although further functional study of the mutation and

genotype-phenotype analysis are necessary, novel compound heterozygous mutations in the IL36RN gene may cause GPP. Sugiura et al. reported that 9 of 11 GPP cases without PV, but only 2 of 20 GPP cases with PV, had mutations in IL36RN [6]. Another study reported IL36RN mutations in patients with GPP-related pustular diseases such as PPP and ACH [10]. In this study, two patients (Nos. 2 and 4) who had preceding typical PV lesions (definite GPP with PV) had no IL36RN mutations, and one patient (No. 8) with preceding ACH had homozygous mutation p.R10X. One (No. 7) of the four patients who did not have preceding skin lesions had heterozygous mutations p.R10X and p.E112K. In this study we carried out sequencing analysis of IL36RN in 8 GPP and 258 PV patients and genotyping analysis of 1130 controls in the Japanese population. We found novel heterozygous mutations, p.R10X and p.E112K, in one GPP patient. Further genetic analysis of the IL36RN gene in GPP and GPP-related pustular diseases together with the underlying mechanism of pustular formation may contribute to the development of specific treatments targeting the IL-36 pathway. Acknowledgments We thank all the individuals who participated in the study. We also thank M.T. Shimizu, H. Sekiguchi, A.I. Jodo, N. Kawaraichi and the technical staff of the Center for Genomic Medicine for providing technical assistance. This work was supported by Health Science Research Grants from the Ministry of Health, Welfare and Labor of Japan and the Ministry of Education, Culture, Sports, Science and Technology, Japan. References

Fig. 1. Direct sequencing of IL36RN gene in GPP patients. (a) Patients 7 and 8 had heterozygous and momozygous c.28C > T mutations in exon 2, respectively. (b) Patient 7 had a heterozygous c.334G > A mutation in exon 5.

[1] 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. [2] 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. [3] 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. [4] Sugiura K, Takeichi T, Kono M, Ogawa Y, Shinomiya Y, Muro Y, et al. A novel IL36RN/ IL1F5 homozygous nonsense mutation, p.Arg10X, in a Japanese patients with adult-onset generalized pustular psoriasis. Br J Dermatol 2012;167:699–701. [5] Farooq M, Nakai H, Fujimoto A, Fujikawa H, Matsuyama A, Kariya N, et al. Mutation analysis of the IL36RN gene in 14 Japanese patients with generalized pustular psoriasis. Hum Mutat 2012;34:176–83. [6] 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. [7] Iwatsuki K, Terui T, Ozawa A, Komine M, Umezawa Y, Torii H, et al. Practice Guidelines 2010 for generalized pustular psoriasis (GPP): treatment guidelines incorporating TNF-a inhibitor. Jpn J Dermatol 2010;120:815–39 [in Japanese].

Letters to the Editor / Journal of Dermatological Science 76 (2014) 255–271 [8] Li M, Han J, Lu Z, Li H, Zhu K, Cheng R, et al. Prevalent and rare mutations in IL-36RN gene in Chinese patients with generalized pustular psoriasis and psoriasis vulgaris. J Invest Dermatol 2013;133:2637–9. [9] 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. [10] 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.

Mitsuha Hayashia,1, Tsuguhisa Nakayamab,c,1, Tomomitsu Hirotac, Hidehisa Saekid,*, Yoshimasa Nobeyamaa, Toshihiro Itoa, Yoshinori Umezawaa, Osamu Fukuchia, Koichi Yanabaa, Sota Kikuchia, Hidemi Nakagawaa, Yuichiro Tsunemie, Sayaka Shibataf, Shinichi Satof, Yayoi Tadag, Akihiko Miyatakeh, Shigeharu Fujiedai, Mayumi Tamaric a Department of Dermatology, The Jikei University School of Medicine, Japan; bDepartment of Otorhinolaryngology, The Jikei University School of Medicine, Japan; cLaboratory for Respiratory and Allergic Diseases, Center for Integrative Medical Sciences, Riken, Japan;

269

d

Department of Dermatology, Nippon Medical School, Japan; Department of Dermatology, Tokyo Women’s Medical University, Japan; fDepartment of Dermatology, Faculty of Medicine, University of Tokyo, Japan; gDepartment of Dermatology, Teikyo University School of Medicine, Japan; hMiyatake Asthma Clinic, Japan; iDepartment of Otorhinolaryngology, University of Fukui Faculty of Medical Sciences, Japan e

*Corresponding author at: Department of Dermatology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Tel.: +81 3 3822 2131; fax: +81 3 3823 6731 E-mail address: [email protected] (H. Saeki). 1

These authors contributed equally to this article.

Received 5 September 2014

http://dx.doi.org/10.1016/j.jdermsci.2014.10.008

Letter to the Editor Ultra-pure soft water improves skin barrier function in children with atopic dermatitis: A randomized, double-blind, placebo-controlled, crossover pilot study

Keywords: Atopic dermatitis; Ultra-pure soft water; Skin barrier function; Out-in skin transparency

Calcium (Ca2+) and magnesium (Mg2+) present in water determine the water hardness. These mineral components react with soap to form an insoluble precipitate known as ‘‘soap scum’’. Since invisible soap scum binds tightly to the skin surface and cannot be rinsed away easily, it might exacerbate atopic dermatitis (AD). In fact, exposure to hard water was reported to be a risk factor for AD in a cohort study of primary school children in Nottinghamshire, UK (where the water hardness is 118–314 mg/ L) [1] and also in Osaka, Japan (35.2–100 mg/L) [2]. To evaluate clinical and objective improvements in skin barrier functions with shower treatment using water devoid of these two mineral components, we used ultra-pure soft water (UPSW), in which Ca2+ and Mg2+ have been almost completely replaced by sodium ions using a cation-exchange resin and we performed a randomized, double-blind, placebo-controlled, crossover pilot study. The CONSORT 2010 flow diagram for this study is shown in Fig. 1. Eligibility criteria for the study were the presence of mild to moderate AD in child patients between 3 and 6 years of age. AD patients who were administered systemic steroid or immunosuppressive therapy or those who had the eczema area and severity index (EASI) score [3] >20 were excluded. The study was approved by the IRB at Chiba University and was registered with the UMIN Clinical Trial Registry (ID: 6136). A machine used to supply either UPSW (Bihadakko; Miura Co., Ltd, Japan) or ordinary tap water without cation-exchange resin (placebo) was set up in the bathroom of each home. Patients were randomly assigned to two groups: 6-week shower treatments with UPSW versus with placebo. After a 2-week washout period using

tap water only, the treatment protocols were switched, and a second 6-week treatment was then performed. Each patient continued using the same therapies and drugs during this study. The baseline and endpoint values of EASI were compared. Transepidermal water loss (TEWL) was assessed using the condenser-chamber AquaFlux1 (Biox System, UK). For out-in skin transparency (OIST), the penetration of tartrazine was measured using the photocolorimeter, as described previously [4]. Pruritus and therapy satisfaction were evaluated by each mother of the patient using the visual analogue scale (VAS). Serum TARC and LDH levels and the eosinophil counts in peripheral blood were measured. The hardness of water was monitored over the course of the study, and that of UPSW kept 1.0 mg/L in this study. The mean changes were analyzed by a repeated measures ANOVA based on the Grizzle model using SAS software version 9.3 (SAS Institute, USA), including effects for treatment, period, and subject sequence with subjects as a random effect. The tests were 2-sided. P < 0.05 was considered to indicate statistical significance. A total of 12 patients were recruited. Group 1 started with UPSW shower treatment contained five patients (one female, mean age  SD: 63.5  5.7 months, EASI  SD: 10.67  5.35, water hardness in tap water  SD: 91.67  38.25 mg/L), while group 2 started with placebo shower treatment contained six patients (three females, age: 52.0  14.2 months, EASI: 7.02  5.15, water hardness: 72.5  9.4 mg/L). One patient withdrew his consent within the first 3 weeks for personal reasons, while the remaining 11 completed the entire course. When compared the mean changes between UPSW and placebo treatment, no significant difference in the EASI was observed (Table 1). For skin barrier function, the TEWL of both abdominal and upper back lesions showed no significant difference, while OIST showed a significant difference between the 2 groups (P = 0.048). In the evaluation of subjective symptoms using the VAS, statistical significance was recognized for pruritus (P = 0.044) and satisfaction with therapy (P = 0.022). As for the laboratory findings assessed, no significant difference in peripheral eosinophil counts or in serum TARC and LDH level were detected between the two groups (data not shown). Other trials investigating water softeners for children with moderate to severe eczema failed to show significant improve-