REPORT Mutations in CSTA, Encoding Cystatin A, Underlie Exfoliative Ichthyosis and Reveal a Role for This Protease Inhibitor in Cell-Cell Adhesion Diana C. Blaydon, 1 Daniela Nitoiu, 1 Katja-Martina Eckl, 2 Rita M. Cabral, 1 Philip Bland, 1 Ingrid Hausser, 3 David A. van Heel, 1 Shefali Rajpopat, 1 Judith Fischer, 4,5 Vinzenz Oji, 6 Alex Zvulunov, 7,8 Heiko Traupe, 6 Hans Christian Hennies, 2,9,10 and David P. Kelsell 1, * Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of non- erythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homo- zygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prom- inent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis. Cystatins form a superfamily of protease inhibitors, which could be further subdivided into three groups on the basis of their different distributions and molecular structure. 1,2 Cystatin A (also known as stefin A) is a mem- ber of the type 1 cystatins (comprising stefin A and B) that have a mainly intracellular localization, although cystatin A has also been detected in sweat and in the medium of cultured keratinocytes. 3 The cystatins are tight, reversible inhibitors of the papain-like lysosomal cysteine proteases, such as cathepsins B, H, and L. 4–6 Furthermore, an imbalance of these proteases and their cystatin inhibitors is found in many cancers and other diseases. 7–9 Cystatin A was originally identified as a component of the cornified cell envelope in the upper layers of the skin 10 and has also been shown to be a potent inhibitor of exogenous proteases, such as the dust mite allergens Der p 1 and Der f 1, 3 which can break down the epidermal barrier of the skin. In addition, cystatin A has been associ- ated with atopic dermatitis (AD [MIM 603165]), 11,12 a chronic inflammatory skin disease often associated with a defective epidermal barrier. 13 We present here a role for cystatin A in the normal physiology of the skin within the lower levels of the epidermis and show that loss-of- function mutations in cystatin A underlie a peeling skin phenotype in the absence of an obvious epidermal barrier defect. We previously reported a large, consanguineous, Bedouin family with autosomal-recessive exfoliative ichthyosis (MIM 607936) presenting in five individuals as circum- scribed peeling of the skin on palms and soles associated with dry and scaly skin (Figures 1A and 1B). Initial linkage analysis with microsatellites showed suggestive linkage to chromosomal region 12q13. 14 In view of the fact that the unaffected mother was uninformative for all the markers in this region of the genome, we decided to reassess the linkage analysis in this family. The study was approved by the South East National Health Service research ethics committee and by the institutional review board of the University Hospital of Mu ¨nster, and all patients enrolled gave their informed consent. High-resolution homozy- gosity mapping was performed by genotyping two affected individuals (Figure 1C, individuals IV-10 and V-3) with the Affymetrix GeneChip Human Mapping 250K Nsp SNP array (Affymetrix, Santa Clara, CA, USA) (see Table S1, avail- able online). GeneChip DNA Analysis Software (Affyme- trix, Santa Clara, CA, USA) was used for calling SNP geno- types and a large block of homozygosity (9333.1 kb), containing approximately 60 genes and shared by both affected individuals, was identified on chromosomal region 3q21 between rs6783609 and rs6438966. Using PLINK to analyze the SNP data, 15 we observed no significant blocks of shared homozygosity on chromosomal region 12q13, and the next largest stretches of homozygosity on 1 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK; 2 Division of Dermatogenetics, Cologne Center for Genomics, University of Cologne, 50931 Cologne, Germany; 3 Department of Dermatology, University Hospital of Heidelberg, 69115 Heidelberg, Germany; 4 Institute of Human Genetics, University Hospital of Freiburg, 79106 Freiburg, Germany; 5 Zentrum fu ¨r Biosystemanalyse, Center for Biological Systems Analysis, University of Freiburg, 79104 Freiburg, Germany; 6 Department of Dermatology, University Hospital of Mu ¨nster, 48149 Mu ¨nster, Germany; 7 Pediatric Dermatology Unit, Schneider Children’s Medical Center, Petah-Tiqva 49100, Israel; 8 Faculty of Health Sciences, Medical School for International Health, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; 9 Cologne Cluster of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Cologne, Germany; 10 Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany *Correspondence: d.p.kelsell@qmul.ac.uk DOI 10.1016/j.ajhg.2011.09.001. Ó2011 by The American Society of Human Genetics. All rights reserved. 564 The American Journal of Human Genetics 89, 564–571, October 7, 2011