New and Recurrent SERPINB7 Mutations in Seven Chinese Patients with Nagashima-Type Palmoplantar Keratosis

J Yin et al. SERPINB7 Mutations in NPPK

end point was to detect new diagnoses of cardiovascular disease, we felt that these exclusion criteria were necessary to calculate the incidence of disease. Patient adherence cannot be assessed from pharmacy records. Other medications, family history, or lifestyle variables were not included in our analysis. All patients who were prescribed a tetracycline, regardless of duration, were included. It is not possible to know whether the prescription was truly given for acne/rosacea versus an infection. There are data that rosacea itself may increase the risk of cardiovascular disease (Duman et al., 2013), and it is possible that any indication for a tetracycline may perhaps positively affect the pro-inflammatory state of the rosacea patient. In private practice, the use of low-dose (20 or 40 mg daily) doxycycline is commonplace in order to take advantage of the sub-antimicrobial dose that also provides an anti-inflammatory effect in rosacea. This formulation is not available at our facility, and most patients were prescribed between 50–200 mg daily. Owing to sampling issues (three different medications in two different groups of patients, acne, and rosacea), we did not analyze the odds ratio for each of the tetracycline medications: tetracycline, doxycycline, and minocycline. Finally, as our veteran population was predominantly male, our results may not be generalizable to a more heterogeneous population.

CONFLICT OF INTEREST JD has received fees from Galderma Laboratories for consulting that was performed after the design and completion of this study.

ACKNOWLEDGMENTS We thank the American Acne and Rosacea Society for their financial support of this study. We acknowledge Tongyu Cao for her assistance in grant proposal, and Robert Kirsner for his encouragement and mentorship. This study was funded by a resident grant from the American Acne and Rosacea Society.

Jacquelyn R. Dosal1, Georgette L. Rodriguez1,2, Candido F. Pezon3, Hua Li4 and Jonette E. Keri1,2 1

Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA; 2Department of Dermatology, Miami Veterans Affairs Health System, Miami, Florida, USA; 3Data Management, Miami Veterans Affairs Health System, Miami, Florida, USA and 4Department of Epidemiology and Public Health, University of Miami Miller School of Medicine, Miami, Florida, USA E-mail: [email protected]

Hackmann AE, Rubin BG, Sanchez LA et al. (2008) A randomized, placebo-controlled trial of doxycycline after endoluminal aneurysm repair. J Vasc Surg 48:519–26 Jackson LA, Smith NL, Heckbert SR et al. (1999) Lack of association between first myocardial infacrtion and past use of erythromycin, tetracycline, or doxycycline. Emerg Infect Dis 5:281–4 Margolis DJ, Hoffstad O, Billker W (2007) Association or lack of association between tetracycline class antibiotics used for acne vulgaris and lupus erythematosus. Br J Dermatol 157:540–6 Margolis DJ, Fanelli M, Hoffstad O et al. (2010) Potential association between the oral tetracycline class of antimicrobials used to treat acne and inflammatory bowel disease. Am J Gastroenterol 105:2610–6 Meier CR (2000) Antibiotics in the prevention and treatment of coronary heart disease. J Infect Dis 181:S558–62 Prodanowich S, Ma F, Taylor JR et al. (2005) Methotrexate reduces incidence of vascular diseases in veterans with psoriasis or rheumatoid arthritis. J Am Acad Dermatol 52:262–7 Romero-Perez D, Fricovsky E, Yamasaki KG et al. (2008) Cardiac uptake of minocycline and mechanisms for in vivo cardioprotection. J Am Coll Cardiol 52:1086–94

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Duman N, Ersoy Evans S, Atakan N (2013) Rosacea and cardiovascular risk factors: a case control study. J Eur Acad Dermatol Venereol (e-pub ahead of print; doi:10.111/jdv.12234)

Sho E, Chu J, Sho M et al. (2004) Continuous perioaortic infusion improves doxycycline efficacy in experimental aortic aneurysms. J Vas Surg 39:1312–21

Griffin MO, Fricovsky E, Ceballos G et al. (2010) Tetracyclines: a pleotropic family of compounds with promising therapeutic properties. Review of the literature. Am J Physiol Cell Pyschiol 299:539–48

Tessone A, Feinberg MS, Barbash IM et al. (2006) Effect of matrix metalloproteninase inhibition by doxycycline on myocardial healing and remodeling after myocardial infarction. Cardivasc Drugs Ther 19:383–90

Griffin MO, Jinno M, Miles LA et al. (2005) Reduction of myocardial infarct size by doxycycline: a role for plasmin inhibition. Mol Cell Biochem 270:1–11

Wang Y, Gao H, Loyd CM et al. (2012) Chronic skin-specific inflammation promotes vascular inflammation and thrombosis. J Invest Dermatol 132:2067–75

See related commentary on pg 2076

New and Recurrent SERPINB7 Mutations in Seven Chinese Patients with Nagashima-Type Palmoplantar Keratosis Journal of Investigative Dermatology (2014) 134, 2269–2272; doi:10.1038/jid.2014.80; published online 10 April 2014

TO THE EDITOR Hereditary palmoplantar keratodermas (PPKs) consist of a heterogeneous group

of hyperkeratotic disorders involving mainly the palms and the soles (Lucker et al., 1994). Nagashima-type palmo-

Abbreviations: NPPK, Nagashima-type palmoplantar keratosis; PPK, palmoplantar keratoderma Accepted article preview online 10 February 2014; published online 10 April 2014

plantar keratosis (NPPK, MIM615598) was initially described in Japanese literature as a mild form of mal de Meleda (MIM248300), which is one of the diffuse autosomal-recessive PPKs (Nagashima, 1977). Later, similar cases were reported both in Japanese and www.jidonline.org 2269

J Yin et al. SERPINB7 Mutations in NPPK

English literature with a proposed name of NPPK (Mitsuhashi et al., 1989; Kabashima et al., 2008). Genetic study of NPPK excluded SLURP1 mutations, which underlie mal de Meleda, indicating that NPPK was a genetically distinct entity (Kabashima et al., 2008). More recently, Kubo et al. (2013) identified biallelic loss-of-function mutations in the SERPINB7 gene, which encodes a member of the serine protease inhibitor (serpin) superfamily, as the genetic basis of NPPK. To date, all the reported patients with NPPK, to our knowledge, are of Japanese origin. Here, we report on seven unrelated Chinese patients with NPPK with four underlying SERPINB7 mutations, of which one is a recurrent variant (c.796C4T), whereas the other three, to our knowledge, are previously unreported. Seven sporadic patients (four males and three females) of Chinese Han ethnicity with diffuse, nonmutilating, and nonepidermolytic PPKs were enrolled. All the patients were unrelated from different provinces of China. No consanguinity or relevant family history was noted in all the cases. The clinical features of the seven patients were summarized in Table 1. In brief, all the patients presented with diffuse, reddish palmoplantar hyperkeratosis that extended to the waists, the Achilles tendons, and dorsum of the hands/feet (Figure 1a, b, and d), with the onset age ranging

from birth to 4 years. Erythrokeratoderma, affecting elbows and knees, was seen in patients 1 and 6 (Figure 1c). Mild hyperhidrosis on the palms and soles, skin maceration, and recurrent tinea pedis were noted in most of the patients. The stratum corneum swelled and exhibited a whitish spongy appearance after the exposure to warm water for 5–10 min (Figure 1d). Histopathological changes are unremarkable, showing orthohyperkeratosis, acanthosis, and mild perivascular inflammatory infiltration of lymphocytes in the upper dermis. Genomic DNA was extracted from peripheral blood of all seven patients and their family members, following their written informed consent. This study was approved by the institution review board in adherence to the Declaration of Helsinki Principles. Sanger sequencing was performed to screen mutations in SERPINB7, SLURP1, and AQP5. AQP5 codes for aquaporin 5 channel and was recently identified as the causative gene of PPK Bothnia-type (Blaydon et al., 2013; Cao et al., 2013), an autosomal-dominant PPK sharing most clinical features with NPPK. No pathogenic mutations were found in SLURP1 or AQP5 in any of the seven patients, whereas four different mutations in SERPINB7 were detected, including one recurrent mutation (c.796C4T, p.R266*) found in 6 patients. Patients 1–4 were homozygous for c.796C4T mutation. In addition to

the heterozygous mutation c.796C4T, patient 5 was found to have a heterozygous frameshift mutation c.650653delCTGT (p.S217Lfs*7), and patient 6 harbored a heterozygous point mutation c.455G4T (p.G152V). The latter causes the change in the first nucleotide of exon 6, leading to an elimination of acceptor-splicing site of this exon (as predicted by BDGP Splice Site Prediction, with a drop in the splicing score from 0.89 to 0.42, see http://www.fruitfly. org/seq_tools/splice.html). Unfortunately, skin biopsy for mRNA verification was unacceptable for patient 6. Patient 7 was homozygous for a frameshift mutation c.522-523insT (p.V175Cfs*46) (Figure 1e-i). Parents of all patients (except patient 5, whose parents were unavailable) were heterozygous carriers of the corresponding mutations, consistent with an autosomal-recessive inheritance in all the families. Two of the mutations identified in this study, c.455G4T and c.796C4T, were found in single nucleotide polymorphism databases. According to the 1000 Genomes Project, c.455G4T (rs202182550) was present in a heterozygous form in 1 of 197 Chinese individuals, and c.796C4T (rs142859678) was found in a heterozygous manner in 6 of 197 Chinese individuals. Allele frequency of these two SNPs adds up to more than 0.017, and the estimated prevalence rate of NPPK is B3 in 10,000 in China.

Table 1. Summary of the clinical features and SERPINB7 mutations of the seven patients Allele 1 Patient

Sex

Allele 2

Age (years) Onset age Nucleotide change Amino-acid change Nucleotide change Amino-acid change

1

Female

25

2

Male

3

Male

4 5

Clinical features in addition to PPK

8 months

c.796C4T

p.R266*

c.796C4T

p.R266*

Mild erythrokeratoderma on the elbows and knees

13

At birth

c.796C4T

p.R266*

c.796C4T

p.R266*

Tinea pedis, hyperhidrosis

17

6 months

c.796C4T

p.R266*

c.796C4T

p.R266*

Tinea pedis, hyperhidrosis

Female

24

6 months

c.796C4T

p.R266*

c.796C4T

p.R266*

Tinea pedis, hyperhidrosis

Male

11

1 year

c.796C4T

p.R266*

c.650-653delCTGT

p.S217Lfs*7

Tinea pedis, hyperhidrosis

6

Female

8

1 year

c.796C4T

p.R266*

c.455G4T

p.G152V and predicted splicing alternation

Mild erythrokeratoderma affecting the elbows and knees, mild pruritus

7

Male

30

4 years

c.522-523insT

p.V175Cfs*46

c.522-523insT

p.V175Cfs*46

Onychomycosis and tinea pedis, hyperhidrosis

Abbreviation: PPK, palmoplantar keratoderma.

2270 Journal of Investigative Dermatology (2014), Volume 134

J Yin et al. SERPINB7 Mutations in NPPK

c.796C>T(p.R266*)

c.796C>T(p.R266*)

c.650-653delCTGT(p.S217Lfs*7)

c.455G>T

c.522-523insT(p.V175Cfs*46)

Figure 1. Clinical features and SERPINB7 mutations detected in patients. (a, b) Bilateral reddish, hyperkeratotic lesions with transgredient erythema and keratosis of the palms and soles. (c) Mild erythrokeratoderma affecting the elbow of patient 1. (d) The whitish and spongy appearance of the stratum corneum in patient 7 after exposure to warm water for 5–10 min. (e) The homozygous mutation c.796C4T identified in SERPINB7 in patients 1–4. (f) The heterozygous mutation c.796C4T identified in SERPINB7 in patients 5 and 6. (g) The heterozygous frameshift mutation c.650-653delCTGT in SERPINB7 in patient 5. (h) The heterozygous mutation c.455 G4T identified in SERPINB7 in patient 6. (i) The homozygous frameshift mutation c.522-523insT identified in SERPINB7 in patient 7.

As mutation c.796C4T was highly prevalent in our patients (Kubo et al., 2013), we employed haplotype analysis in the 6 patients harboring mutation c.796C4T to determine whether this mutation was derived from one common ancestor or it represented a mutation hotspot. Ten SNPs that are highly polymorphic in Chinese populations adjacent to the mutation were chosen and genotyped by Sanger sequencing. Patients 1–4 shared identical homozygous alleles in all 10 SNPs, and patients 5 and 6 harbored all the alleles of the other four patients (Supplementary Table S1 online), suggesting that these six patients are likely to share a

common mutant haplotype. Therefore, it is reasonable to infer that, instead of a mutation hotspot, c.796C4T was a founder mutation. Similar founder effect was also demonstrated in TGM5, in which a recurrent mutation c.337G4T, with an allele frequency of 0.015 in Northern Europeans, is commonly present in European patients with acral peeling skin syndrome (Pigors et al., 2012; van der Velden et al., 2012). SERPINB7 encodes a subtype of clade-B serpins which inhibit serine proteases and protect cells from proteasemediated damage (Silverman et al., 2004). SERPINB7 was distributed in the epidermis throughout the body,

especially in the stratum granulosum and upper part of the stratum corneum. Loss-of-function of SERPINB7 may induce overactivation of proteases specifically in NPPK skin lesions, resulting in protein degradation of keratinocytes, and facilitating water permeation into the stratum corneum (Kubo et al., 2013). All of the truncated proteins of SERPINB7 in our patients were predicted to lose the entire motif of the reactive site loop, which is located at amino acids 331–352 and crucial for its function (Kubo et al., 2013), leading to a complete absence of the protease inhibitory activity of SERPINB7. www.jidonline.org 2271

DJ Margolis et al. Exome Sequencing of Filaggrin and Related Genes

In conclusion, we ascertained seven Chinese individuals with NPPK and identified four causative mutations in SERPINB7, two of which were polymorphic present in normal Chinese individuals. NPPK was estimated to be one of the most common types of autosomalrecessive PPKs in China despite its delayed recognition. Moreover, we confirmed that the recurrent nonsense mutation c.796C4T, prevalent both in Chinese and Japanese NPPK patients, probably descended from the same ancestor. Finally, mutation screening of c.796C4T in SERPINB7 can serve as a cost-effective diagnostic strategy in the setting of Chinese and Japanese patients with diffuse, nonmutilating-inherited PPKs, but without distinctive clinical or histopathological features.

National Natural Science Foundation of China (grant nos. 81201220 and 81271744).

Jinghua Yin1,2, Guiwen Xu1,2,3, Huijun Wang1,2,4, Jiahui Zhao1,2, Lina Duo1,2,4, Xu Cao1,2,4, Zhanli Tang3, Zhimiao Lin1,2 and Yong Yang1,2,4 1

Department of Dermatology, Peking University First Hospital, Beijing, China; 2Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Beijing, China; 3Department of Dermatology, The Affiliated Hospital of Qingdao University, Qingdao, China and 4 Peking-Tsinghua Center for Life Sciences, Beijing, China E-mail: [email protected] or [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We are grateful to the patients and family members who participated in this study. We thank Ming Yang Lee, Madelaine Niam, and Sindhu Subramaniam at University College London for the final proofreading. This work was supported by the

Blaydon DC, Lind LK, Plagnol V et al. (2013) Mutations in AQP5, encoding a water-channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma. Am J Hum Genet 93:330–5 Cao X, Yin J, Wang H et al. (2013) Mutation in AQP5, encoding aquaporin 5, causes palmoplantar keratoderma Bothnia type. J Invest Dermatol 134:284–7

Kabashima K, Sakabe J, Tokura Y et al. (2008) ‘‘Nagashima-type’’ keratosis as a novel entity in the palmoplantar keratoderma category. Arch Dermatol 144:375–9 Kubo A, Shiohama A, Sasaki T et al. (2013) Mutations in SERPINB7, encoding a member of the serine protease inhibitor superfamily, cause Nagashima-type palmoplantar keratosis. Am J Hum Genet 93:945–56 Lucker GP, Kerkhof PC, Steijlen PM (1994) The hereditary palmoplantar keratoses: an updated review and classification. Br J Dermatol 131:1–14 Mitsuhashi Y, Hashimoto I, Takahashi M (1989) Meleda type keratosis palmoplantaris (Nagashima). Practical Dermatol 11:297–300 (in Japanese) Nagashima M (1977) Palmoplantar keratoses. Jinrui Idengaku Shosho 9:23–7 (in Japanese) Pigors M, Kiritsi D, Cobzaru C et al. (2012) TGM5 mutations impact epidermal differentiation in acral peeling skin syndrome. J Invest Dermatol 132:2422–9 Silverman GA, Whisstock JC, Askew DJ et al. (2004) Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis. Cell Mo Life Sci 61:301–25 van der Velden J, Jonkman MF, McLean WHI et al. (2012) A recurrent mutation in the TGM5 gene in European patients with acral peeling skin syndrome. J Derma Sci 65:74–6

Exome Sequencing of Filaggrin and Related Genes in African-American Children with Atopic Dermatitis Journal of Investigative Dermatology (2014) 134, 2272–2274; doi:10.1038/jid.2014.126; published online 10 April 2014

TO THE EDITOR Atopic dermatitis (AD) is a common chronic relapsing disease. There is a considerable body of evidence supporting a genetic basis for AD (Bussman et al., 2011; Ellinghaus et al., 2013). Mutations in the Filaggrin (FLG) gene have been consistently found to be associated with AD in people of European and Asian ancestry (Brown and McLean, 2012). More than 40 FLG loss-of-function mutations have been

described in Europeans and Asians (Brown and McLean, 2012). However, FLG loss-of-function mutations have not commonly been found in Africans or African Americans (Winge et al., 2011a; Brown and McLean, 2012; Margolis et al., 2012). Loss-of-function mutations in exon 3 of FLG result in diminished or absent filaggrin protein, most often due to a premature stop codon or a frameshift mutation resulting in a stop codon further downstream. Interestingly, the

Abbreviations: AD, atopic dermatitis; FLG, Filaggrin; MAF, minor allelic frequency; SFTP, S100-fused-like protein Accepted article preview online 7 March 2014; published online 10 April 2014

2272 Journal of Investigative Dermatology (2014), Volume 134

absence of profilaggrin protein (precursor of filaggrin) has also been noted in keratohyalin granules in the majority of individuals with ichthyosis vulgaris of European and Asian ancestry (Fleckman and Brumbaugh, 2002; Perusquia-Ortiz et al., 2013; Thyssen et al., 2013). FLG is located on chromosome 1q21 in a region called the epidermal differentiation complex. It is part of a family of genes that code for S100-fused-like proteins (SFTP). The SFTPs include the proteins profilaggrin (coded by FLG), hornerin (HRNR), filaggrin-2 (FLG2), repetin (RPTN), cornulin (CRNN), trichohyalin (TCHH), and trichohyalin-like 1 (TCHHL1; Henry et al., 2012). These