Allen 2001 Netherton A2. Netherton Netherton

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3 Netherton Netherton Magert 1999 LEKTI Chavanas 2000 SPINK5 Netherton SPINK5 5p31-32 LEKTI Netherton Netherton Netherton Allen 2001 Netherton SPINK5 A2-1 Netherton Netherton Netherton Netherton Netherton skin-surface biopsy SPINK5 Netherton SPINK5 LEKTI skin-surface biopsy Netherton syndrome (NS) is a severe autosomal recessive ichthyosis. It is characterized by congenital ichthyosiform erythroderma (CIE), trichorrhexis invaginata (TI), which is a distinctive hair-shaft anomaly, and atopic diathesis. Recently, pathogenic mutations in SPINK5 have been identified in NS. SPINK5 encodes lympho-epithelial Kazal-type-related inhibitor

4 (LEKTI) which is a new type of serine protease inhibitor involved in the regulation of skin barrier formation and immunity. Here we study the mutation analysis of four Taiwanese family with NS manifesting CIE with pathognomic ichthyosis linearis circumflexa and TI. Direct DNA sequencing of SPINK5 demonstrated a compound heterozygous mutation in the first case, 2260A T (K754X) in exon 24 and 2468delA in exon 26. The former mutation is a novel mutation and was detected in the mother. The latter mutation was detected in the father and has been previously reported in several European families. Both mutations are expected to result in premature termination codons. In the second case, a homozygous 2260AT (K754X) mutation in exon 24. Mutation analysis could provide a reliable prenatal diagnosis of this lethal ichthyosis in the subsequent pregnancies of such couples. Ultrastructural study of the stratum corneum revealed premature degradation of corneodesomosomes (CDs). Stratum corneum hydrolytic enzymes are responsible for normal desquamation by degrading superficial CDs. Exfoliative toxin A produced by S. aureus is a serine protease that cleaves desmoglein 1 which is an important adhesive molecule of corneodesmosomes (CDs). Premature degradation of CDs in our patients might be attributable in part to insufficient LEKTI to inhibit stratum corneum hydrolytic enzymes and to the exotoxin-producing S. aureus infections or colonization. Further study is needed to prove this hypothesis. Key Words: Netherton syndrome, SPINK5 gene, LEKTI, skin barrier function, serine protease inhibitor, skin surface biopsy, Netherton syndrome (NS) is a rare autosomal recessive disorder of cornification, characterized by the triad of ichthyosis, hair shaft defects, and atopy. The infant with NS typically displays a generalized erythroderma covered by fine, translucent scales. Some infants with NS develop progressive hypernatremic dehydration, failure to thrive, and enteropathy. The stratum corneum (SC) is the outermost layer of the stratified squamous epithelium lining the body surface. It has important physicochemical barrier functions and is the main barrier to water losses through the skin. The mechanical resistance of the SC is dependent on the cohesion between individual corneocytes, which is mediated to a major extent by modified desmosomes. There is balanced to give a constant and well-regulated SC thickness by cell shedding at the skin surface in a process called desquamation. An imbalance between production of the SC and the rate of desquamation may lead to some diseases such as ichthyosis and NS. Recent works has established that proteolysis of intercellular cohesion structures is a major importance in the series of events preceding desquamation. Desmosomes are digested by proteases like plasmin during keratinocytes dissociation. To control the action of proteinase in vivo, organisms produce another group of proteins, namely the proteinase inhibitors.

5 LEKTI (Lympho-epithelial-kazal-type-related-inhibitor) is a new type of human serine protease inhibitor recently identified by Mägert et al. (1999). The entire precursor protein of LEKTI, as deduced from the nucleotide sequence of the cloned cdna, includes 15 potential inhibitory domains, 13 of which exhibit a Kazal-type-derived 4-cysteine-residue pattern that represents a novel protein module of serine protease inhibitor. One of which was found to exert an anti-trypsin activity in vitro. SPINK5, the gene encoding LEKTI, has been linked to 5p31-32, is expressed in epithelial and mucosal surfaces and in the thymus. LEKTI was the first serine-protease inhibitor shown to be primarily involved in a skin disorder or hair morphogenesis, namely, NS. Underscored the importance of the regulation of proteolysis in epithelia formation and keratinocyte terminal differentiation. Incompletely processed lipids and disorganized lamellae, as well as deposits of amorphous electron-dense materials surround the corneocytes in NS. These abnormalities could explain the impaired permeability barrier in NS, and account for hypernatremia and dehydration in infants with NS. The barrier functions, however, have not been directly measured in NS. Allen et al., 2001, have proposed the pathogenetic mechanism of the skin lesion in NS. Genetic defect in SPINK5 may lead to premature activation of the SC tryptic enzyme (SCTE), which would result in activation of phospholipase A2. This may stimulate premature lamellar body secretion as well as disruption of the plasma membrane, triggering cytolysis or premature cornification. At the same time, unchecked activation of the SCTE would lead to premature degradation of corneodesmosomes, as well as premature desquamation and thinning of the SC. Finally, serine proteases may also be involved in releasing SC interleukin 1 from its inactive form, which could lead to the marked inflammation characteristic of NS. Recent advance has implicated serine proteinase inhibitors as key regulatory enzyme in the development and progression of both chronic and acute allergic, inflammatory, and immunological diseases, and as a potentially important target for therapeutic intervention. In this study, we will perform mutation analysis of SPINK5 gene in NS patients, check the barrier function of NS, and administer different serine protease inhibitors, zinc sulfate, tacrolimus ointment and vaseline by topical application to patients with NS to assess their ability to reduce scaling, inflammation and the recovery of skin barrier function. Use skin-surface biopsy and transmission electron microscope examination to evaluate the effect of serine protease inhibitors on the maturation of SC. A. Mutation analysis Mutation analysis: In each family, the entire coding sequence of SPINK5 and flanking intron boundaries are amplified and screened for mutations by conformation-sensitive gel

6 electrophoresis (CSGE) analysis, followed by direct DNA sequencing and verification of mutations. PCR amplification of SPINK5 coding sequence The PCR products were examined on 2% agarose gels and purified by QIAquick columns (Qiagen, Chatsworth, USA), followed by direct DNA sequencing with an ABI 377 automatic sequencer (Advanced Biotechnologies, Columbia, MD, U.S.A.) with both forward and backward primers. B. Clinical evaluation of skin lesions and barrier functions of NS patients * Clinical evaluation * Assessment of barrier functions - Transepidermal water loss (TEWL) - Water content (hydration, skin capacitance) - Sebum content C. Effect of topical serine protease inhibitors (SPI) * Evaluation of the skin lesions improvement * Assessment of barrier functions recovery * Electron microscopy 1. Netherton syndrome J Formos Med Assoc Jun;102(6): (A compound heterozygous mutation of the SPINK5 gene in a Taiwanese boy with Netherton syndrome) Netherton syndrome 4. skin surface biopsy electron microscopy non-invasive stratum corneum 5. protease inhibitorskin surface biopsy electron microscopy Netherton syndrom

7 heat, sweating S. aureus ETA-producing S. aureus ETA (serine protease) Colonization Cleavage of dsg1 & corneodesmosome Overt infection Cleavage of dsg1 in desmosome Cleavaged dsg1 in granular layer Exfoliation of whole horney layer Ceramide deficiency Ceramidase AD S. aureus