LETTERS TO THE EDITOR
Mutations in the c-Secretase Genes NCSTN, PSENEN, and PSEN1 Underlie Rare Forms of Hidradenitis Suppurativa (Acne Inversa) Journal of Investigative Dermatology (2012) 132, 2459–2461; doi:10.1038/jid.2012.162; published online 24 May 2012
TO THE EDITOR Hidradenitis suppurativa (HS; OMIM1 42690) is a chronic inflammatory skin disease that presents with nodules, cysts, and abscesses in apocrine gland–bearing sites. It affects 1% of Europeans and is primarily thought to be a disease of follicular occlusion (Alikhan et al., 2009). Associated factors include smoking (B90%), obesity (475%), and infection (Alikhan et al., 2009). HS may segregate as an autosomal dominant trait, and heterozygous mutations in the g-secretase genes NCSTN, PSENEN, and PSEN1 have recently been reported in a small number of kindreds (Wang et al., 2010; Li et al., 2011; Liu et al., 2011; Pink et al., 2011). We previously identified mutations in NCSTN and PSENEN in two of seven multiplex HS pedigrees. In contrast to previous studies, which have predominantly focussed on the identification of mutations in multiplex kindreds, the aim of this study was to determine the prevalence of mutations in each of these genes among a large cohort of subjects sequentially recruited from a tertiary referral HS clinic. We performed mutational analysis of NCSTN, PSENEN, and PSEN1 by direct sequencing of all coding regions and assessment of large-scale deletions and duplications by multiplex ligation phosphorylation assay (MLPA). In all, 48 individuals with HS (diagnostic criteria from Von der Werth et al., 2000) were sequentially consented and recruited from our tertiary referral clinic. The study was conducted in accordance with the Declaration of
Helsinki Principles and approved by the East London REC2 (09/H0704/50). Population data are shown in Supplementary Table S1 online. The cohort was of mixed ethnicity, the average body mass index (BMI) was 31.1 (clinically obese), 69% reported a smoking history, and the average Sartorius score (Sartorius et al., 2003) was 49.9. A total of 20 (42%) patients reported a family history of HS, 19 consistent with autosomal dominant inheritance. The DNA was extracted from venous blood or saliva. All coding regions and associated splice sites of NCSTN, PSEN1, and PSENEN were amplified by PCR, using exon flanking intronic primers and Sanger sequenced in all 48 patients. In three subjects we identified heterozygous DNA changes in NCSTN (Supplementary Table S2 online), a missense substitution (NCSTN c.553G4A, p.Asp185Asn), and two single-base substitutions located within 10 bp of donor splice junctions (NCSTN c.996 þ 7G4A and NCSTN c.1101 þ 10A4G). To our knowledge these variants have not previously been reported and none were observed in dbSNP, 1,000 genomes, or in 400 European controls. All 48 patients were assessed for whole gene/exon deletions and duplications in NCSTN, PSENEN, and PSEN1 using MLPA (Supplementary Methods online; Hills et al., 2010). No genomic deletions or duplications were detected. The heterozygous missense variant in exon 5 of NCSTN (c.553G4A, p.Asp185Asn) was identified in a 45-yearold female (patient HS-01; Supplementary Table S2 online, Figure 1). The
Abbreviations: BMI, body mass index; HS, hidradenitis suppurativa; MLPA, multiplex ligation phosphorylation assay
& 2012 The Society for Investigative Dermatology
missense variant is predicted to lead to the substitution of an evolutionary conserved aspartic acid residue with an asparagine residue. A heterozygous substitution of the conserved seventh base of intron 8 of NCSTN (c.996 þ 7G4A) was identified in a 38-year-old female (patient HS-02 Supplementary Table S2 online, Figure 1). Computational splice site analysis (https://splice. uwo.ca) predicts this substitution to have a detrimental effect on splicing. NCSTN mRNA expression levels were significantly lower than wild-type controls, suggesting that the mutant transcript is subject to nonsense-mediated decay (Supplementary Methods and Supplementary Figure S1 online). A third heterozygous variant was detected in the donor splice site of NCSTN exon 9 (c.1101 þ 10A4G) in a 24-year-old male (patient HS-03; Supplementary Table S2 online, Figure 1). Computational splice site analysis suggests that this mutation is unlikely to have a significant effect on splicing. Indeed, no aberrant transcripts were detected (via reverse transcriptase–PCR and sequencing of the full-length NCSTN transcript, Supplementary Figures S2 and S3 online) and NCSTN mRNA expression level was within the range of wild-type controls (data not shown). The potential pathogenicity of this variant is therefore unclear and it may represent a rare variant unlinked to HS in this individual. The two individuals with likely pathogenic variants in NCSTN had chronic, severe disease (Sartorius scores of 56 and 160, respectively) comprising painful nodules, cysts, abscesses, sinus tracts, and scarring, involving at least the axillae, groin, and buttocks (see Supplementary Table S2 online). Their www.jidonline.org 2459
AE Pink et al. g-Secretase Mutations in HS
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Figure 1. Three individuals with variants in NCSTN (HS-01, HS-02, and HS-03). (a) Patient HS-01. Inflammatory nodules, abscesses, and scarring in the sub-mammary region. Arrow indicates the NCSTN c.553 G4A variant, a heterozygous substitution in exon 5 of NCSTN (Genbank accession number: NM_015331). (b) Patient HS-02. Inflammatory nodules and extensive scarring in the axilla. Arrow indicates the NCSTN c.996 þ 7 G4A variant, a heterozygous substitution of the seventh base of the donor splice site within intron 8 of NCSTN. (c) Patient HS-03. Inflammatory nodules, sinus tracts, and scarring in the groin. Arrow indicates the NCSTN c.1101 þ 10 A4G variant, a heterozygous substitution of the tenth base of the donor splice site within intron 9 of NCSTN.
Pink et al., 2011 and current study Wang et al., 2010 Li et al., 2011 Liu et al., 2011
Missense mutations
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Figure 2. The location of all reported mutations within the c-secretase complex in HS. Mutations are represented as stars. Both of the missense variants reported to date (p.Asp185Asn (patient HS-01) and the previously reported p.Pro211Arg (Li et al., 2011)) are located within the ectodomain of nicastrin.
2460 Journal of Investigative Dermatology (2012), Volume 132
disease started at the age of 13 and 35 years, respectively. They had a raised BMI (38 and 37) and were both current smokers. Both reported a history of type II diabetes, neither had acne vulgaris, and there was no history of inflammatory bowel disease or other neutrophilic dermatoses. The only effective treatment option was surgical resection (see Supplementary Table S2 online). Interestingly, neither of these individuals reported a family history of HS or any associated conditions. g-Secretase is an endoprotease complex involved in the intramembranous cleavage of numerous type-1 transmembrane proteins. To date, mutations have been reported in three of the six genes encoding proteins integral to the complex (eight in NCSTN, three in PSENEN, and one in PSEN1, Figure 2) (De Strooper et al., 1999; Wang et al., 2010; Li et al., 2011; Liu et al., 2011; Pink et al., 2011). All but one of these mutations were observed in multiplex kindreds, whereas the HS cohort presented here is representative of patients recruited from a tertiary referral clinic (only 42% of patients reported a family history). Our data suggest that NCSTN, PSEN1, and PSENEN are only responsible for a small proportion of HS cases (o7%), and this figure may be lower in the general disease population given the phenotypic severity of the cohort studied. Of the 14 potential g-secretase mutations now reported in HS, 2 are nonsense mutations, 6 result in frameshifts, 4 in altered splicing, and 2 are missense mutations. The pattern of mutations suggests that loss-of-function of components of the g-secretase complex underlies the disease. Interestingly, the NCSTN missense mutation reported here (p.Asp185Asn) and the previously reported p.Pro211Arg (Li et al., 2011) are both located within the ectodomain of NCSTN (Figure 2). These observations support a key functional role for this domain within the g-secretase complex and a critical role in HS pathogenesis. It is currently difficult to draw robust genotype–phenotype correlations; however, the clinical phenotype of all reported mutation-positive cases is severe and extensive. In summary, we examined the gsecretase genes NCSTN, PSENEN, and
D Suzuki and M Senoo p63 Phosphorylation in Epidermal Stem Cells
PSEN1 for mutations in 48 individuals with HS and identified three variants in coding regions and splice sites of NCSTN. To our knowledge these variants are previously unreported. We present evidence consistent with pathogenicity of two of these alleles, adding to the spectrum of described mutations in this gene. Neither individual in whom mutations were identified reported a family history of HS demonstrating the importance of NCSTN in sporadic disease. These data confirm that germline mutations of g-secretase are confined to a minority of subjects who develop HS and suggest that additional and as yet unknown genes predispose to the development of this distressing disorder. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We express our gratitude to the patients for participating in this study. We also acknowledge support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre
award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. AEP is supported by a Medical Research Council/British Association of Dermatologists/ British Skin Foundation Clinical Research Training Fellowship. We thank Kate Thornberry, Naomi Hare, Michael Allen, and Robert Pleass (Skin Therapy Research Unit, St John’s Institute of Dermatology, Guy’s and St Thomas’ NHS Foundation Trust, King’s College London) for their contribution to this work.
Andrew E. Pink1, Michael A. Simpson1, Nemesha Desai2, Dimitra Dafou1, Alison Hills3, Peter S. Mortimer4, Catherine H. Smith2, Richard C. Trembath1,5 and Jonathan N.W. Barker1 1 Division of Genetics and Molecular Medicine, King’s College London School of Medicine, London, UK; 2St John’s Institute of Dermatology, Guy’s & St Thomas’ NHS Foundation Trust, London, UK; 3Department of Cytogenetics, GSTS Pathology, London, UK; 4 Division of Clinical Science, St George’s University of London, London, UK and 5 Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK E-mail:
[email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Alikhan A, Lynch PJ, Eisen DB (2009) Hidradenitis suppurativa: a comprehensive review. J Am Acad Dermatol 60:539–61 De Strooper B, Annaert W, Cupers P et al. (1999) A presenilin-1-dependent gamma-secretaselike protease mediates release of Notch intracellular domain. Nature 398:518–22 Hills A, Ahn JW, Donaghue C et al. (2010) MLPA for confirmation of array CGH results and determination of inheritance. Mol Cytogenet 3:19 Li CR, Jiang DB, Shen DB et al. (2011) Two novel mutations of the nicastrin gene in Chinese patients with acne inversa. Br J Dermatol 165:415–8 Liu Y, Gao M, Lv YM et al. (2011) Confirmation by exome sequencing of the pathogenic role of NCSTN mutations in acne inversa (hidradenitis suppurativa). J Invest Dermatol 131:1570–2 Pink AE, Simpson MA, Brice GW et al. (2011) PSENEN and NCSTN mutations in familial hidradenitis suppurativa (acne inversa). J Invest Dermatol 131:1568–70 Sartorius K, Lapins J, Emtestam L et al. (2003) Suggestions for uniform outcome variables when reporting treatment effects in hidradenitis suppurativa. Br J Dermatol 149:211–3 Von der Werth JM, Williams HC, Raeburn JA (2000) The clinical genetics of hidradenitis suppurativa revisited. Br J Dermatol 142:947–53 Wang B, Yang W, Wen W et al. (2010) Gammasecretase gene mutations in familial acne inversa. Science 330:1065
Increased p63 Phosphorylation Marks Early Transition of Epidermal Stem Cells to Progenitors Journal of Investigative Dermatology (2012) 132, 2461–2464; doi:10.1038/jid.2012.165; published online 24 May 2012
TO THE EDITOR The mammalian epidermis is maintained by epithelial stem cells (SCs) capable of self-renewal and differentiation (Fuchs, 2009). Although the transcription factor p63 is critical for the proliferative potential of epithelial SCs (Senoo et al., 2007), the molecular events underlying their transition to more differentiated progenitors remain largely unknown. Technical challenges in distinguishing SCs from transit-amplifying (TA) cells, early progeny of SCs (Watt, 2001),
complicate such analyses and impede development of new strategies to improve SC quality for therapeutic transplantation and SC-directed gene therapy. A current model dictates that SCs in the interfollicular epidermis are located in the basal layer, whereas TA cells are found in both the basal and suprabasal layers (Watt, 2001; Fuchs, 2008). As epidermal SCs express relatively high levels of p63 (p63hi) in culture (Pellegrini et al., 2001; Senoo et al., 2007), we first investigated whether p63hi cells were
Abbreviations: CK14, cytokeratin-14; HPEK, human primary epidermal keratinocyte; SC, stem cell; TA, transit amplifying
restricted to the epidermal basal layer. To identify the basal layer, we used integrin b4 (Radoja et al., 2006). Unexpectedly, we found p63hi cells in both basal and suprabasal layers in adult human epidermis (Figure 1a and b), suggesting that high p63 expression is not restricted to SCs. Because the decrease in p63 expression accompanying epidermal cell differentiation appears linked to phosphorylation and consequent targeting for proteasomemediated degradation (Westfall et al., 2005), we next asked whether initiation of SC differentiation is accompanied by an increase in p63 phosphorylation. www.jidonline.org 2461
