A novel SPINK5 donor splice site variant in a child with Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a genodermatosis caused by loss-of-function mutations in SPINK5, resulting in aberrant LEKTI expression. METHOD: Next-generation sequencing of SPINK5 (NM_001127698.1) was carried out and functional studies were performed by immunofluorescence microscopy of a lesional skin biopsy using anti-LEKTI antibodies. RESULTS: We describe a novel SPINK5 likely pathogenic donor splice site variant (NM_001127698.1:c.2015+5G>A) in a patient with NS and confirm its functional significance by demonstrating complete loss of LEKTI expression in lesional skin by immunofluorescence analysis. CONCLUSION: The 2015+5G>A is a novel, likely pathogenic variant in NS. Herein we review and assimilate documented SPINK5 pathogenic variants and discuss possible genotype-phenotype associations in NS.

Genetic activation of Nrf2 reduces cutaneous symptoms in a murine model of Netherton syndrome.

Netherton syndrome is a monogenic autosomal recessive disorder primarily characterized by the detachment of the uppermost layer of the epidermis, the stratum corneum It results from mutations in the SPINK5 gene, which codes for a kallikrein inhibitor. Uncontrolled kallikrein activity leads to premature desquamation, resulting in a severe epidermal barrier defect and subsequent life-threatening systemic infections and chronic cutaneous inflammation. Here, we show that genetic activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2/Nrf2) in keratinocytes of Spink5 knockout mice, a model for Netherton syndrome, significantly alleviates their cutaneous phenotype. Nrf2 activation promoted attachment of the stratum corneum and concomitant epidermal barrier function, and reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α and thymic stromal lymphopoietin. Mechanistically, we show that Nrf2 activation induces overexpression of secretory leukocyte protease inhibitor (Slpi), a known inhibitor of kallikrein 7 and elastase 2, in mouse and human keratinocytes in vivo and in vitro, respectively. In the Spink5-deficient epidermis, the upregulation of Slpi is likely to promote stabilization of corneodesmosomes, thereby preventing premature desquamation. Our results suggest pharmacological NRF2 activation as a promising treatment modality for Netherton syndrome patients.This article has an associated First Person interview with the first author of the paper.

A proinflammatory role of KLK6 protease in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare but severe type of ichthyosis characterized by atopy, allergies, and potentially lethal skin overdesquamation associated with highly elevated proteolytic activities in LEKTI-deficient epidermis. NS symptoms are recapitulated in Spink5-/- mouse where the gene encoding Lekti has been invalidated. Spink5-/- mice die within 5h from birth due to their severe skin barrier defect leading to dehydration. Spink5-/- mice also serve as a model for atopic dermatitis. The KLK6 protease is expressed by epidermal keratinocytes and shown in vitro to cleave desmosomal components. OBJECTIVE: To investigate in vivo whether KLK6 is implicated in epidermal overdesquamation and/or inflammation associated with NS. METHODS: The role of KLK6 was evaluated by generating Spink5-/-Klk6-/- double knockout mice. The phenotype was assessed by macroscopic observation, immunohistochemistry for differentiation markers, in situ zymography for proteolysis, and quantification of proinflammatory cytokines. RESULTS: Elimination of Klk6 in Spink5-/- remarkably suppresses the expression of Tslp, a major itching-inducing factor and driver of allergic reactions. Tnfα and the Th17 promoting cytokine Il-23 were also suppressed. Spink5-/-Klk6-/- mice display normalized keratinocyte differentiation, nevertheless, epidermal proteolytic activities and the associated overdesquamation were not ameliorated, and Spink5-/-Klk6-/- still died from a severe epidermal barrier defect as the Spink5-/-. CONCLUSIONS: Ablation of Klk6 largely suppresses epidermal inflammation but cannot rescue overdesquamation leading to the lethal NS phenotype. Nonetheless, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other inflammatory conditions e.g. atopic dermatitis.

Toll-like receptor signaling induces the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes.

BACKGROUND: Lympho-epithelial Kazal-type inhibitor (LEKTI) tightly controls the activities of serine proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 in the epidermis. LEKTI is known to be an essential molecule for the epidermal skin barrier, as demonstrated by SPINK5 nonsense mutation, which results in Netherton syndrome. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and produce inflammatory cytokines, chemokines, and antimicrobial peptides. However, the effect of TLR signaling on the expression of LEKTI is not clear. OBJECTIVE: To investigate whether TLR signaling can affect expression of LEKTI in epidermal keratinocytes. METHODS: We stimulated a panel of TLR ligands and investigated the expression of LEKTI in normal human epidermal keratinocytes (NHEKs). We further measured trypsin or chymotrypsin-like serine protease activity in NHEK cultured media under stimulation with TLR3 ligand, poly (I:C). Immunostaining for LEKTI was performed using skin samples from skin infectious diseases. RESULTS: TLR1/2, 3, 5, and 2/6 ligands induced the expression of LEKTI in NHEKs. The trypsin or chymotrypsin-like serine protease activity in NHEKs was up-regulated with the stimulation of poly (I:C). The gene expressions of KLK6, KLK10, KLK11, and KLK13 were also increased by poly (I:C). An immunohistochemical analysis demonstrated that the expression of LEKTI was up-regulated in the lesions of varicella, pyoderma, and rosacea. CONCLUSIONS: TLR signaling induces the expression of LEKTI in epidermal keratinocytes, which might contribute to the control of aberrant serine protease activities in inflammatory skin diseases.

Skin Biopsy in Netherton Syndrome: A Histological Review of a Large Series and New Findings.

Netherton syndrome (NS) is a severe genetic skin disorder, with often delayed or misleading clinical signs. The histological features of skin biopsies, usually described as a psoriasiform hyperplasia, have only been reported in isolated case reports or small case series. The aim of this study is to define, for the first time, the precise histological pattern of cutaneous lesions, in a large cohort of skin biopsies from confirmed NS patients. The study included 80 consecutive skin biopsies from 67 patients taken between January 1995 and June 2014. All were from confirmed NS patients with either a negative lympho-epithelial Kazal-type-related inhibitor (LEKTI) immunohistochemistry and/or molecular confirmation by identified mutation in SPINK5. In this cohort, the most frequent histological finding was also psoriasiform hyperplasia, but there were additional, less common, or previously unreported findings, including compact parakeratosis with large nuclei, subcorneum or intracorneum splitting, presence of clear cells in the upper epidermis or stratum corneum, dyskeratosis, dermal infiltrate with neutrophils and/or eosinophils, and dilated blood vessels in the superficial dermis. An early confirmation of the diagnosis of NS is essential for improved patient management. Thus, in the situation of a patient with an unknown skin disorder and non specific clinical presentation, the dermatopathologist may now be able to suggest the diagnosis of NS based on these newly reported characteristics. However, LEKTI immunohistochemistry remains the essential diagnostic investigation in cases with misleading or nonspecific histological features and is mandatory for the definitive diagnosis of NS in all patients.

ICHTHYOSIS LINEARIS CIRCUMFLEXA: NOT ALWAYS A PART OF NETHERTON SYNDROME.

Ichthyosis Linearis Circumfiexa (ILC) is a rarely seen autosomal recessive keratinization disorder and is characterized by erythematous, polycystic, plaques with 'double-edged' scales. Its histological features resemble psoriasis. A triad of ichthyosis (usually Ichthyosis Linearis Circumfiexa), trichorrhexis invaginata (a distinctive hair shaft disorder) and atopy is named as Netherton syndrome. Herein, we report a 12 year-old girl presenting with ILC not accompanied with typical atopy findings and hair shaft disorder.

Trichoscopy as a diagnostic tool in trichorrhexis invaginata and Netherton syndrome

Netherton syndrome is a rare autosomal recessive disease characterized by erythroderma, ichthyosis linearis circumflexa, atopy, failure to thrive and a specific hair shaft abnormality called trichorrhexis invaginata or bamboo hair, considered pathognomonic. We report the case of a 4-year-old boy with erythroderma since birth, growth deficit and chronic diarrhea. Trichoscopy was used to visualize typical bamboo and "golf tee" hair and of key importance to diagnose Netherton syndrome. We suggest the use of this procedure in all children diagnosed with erythroderma.

Epidermal barrier abnormalities in exfoliative ichthyosis with a novel homozygous loss-of-function mutation in CSTA.

Autosomal recessive exfoliative ichthyosis (AREI) results from mutations in CSTA, encoding cysteine protease inhibitor A (cystatin A). We present a 25-year-old man from Iran with consanguineous parents, who presented with congenital erythroderma, hyperhidrosis and diffuse hyperkeratosis with coarse palmoplantar peeling of the skin, aggravated by exposure to water and by occlusion. Candidate gene analysis revealed a previously unknown homozygous loss-of-function mutation c.172C>T (p.Arg58Ter) in CSTA, and immunostaining showed absence of epidermal cystatin A, confirming the diagnosis of AREI. Ultrastructural analysis by transmission electron microscopy showed normal degradation of corneodesmosomes, mild intercellular oedema in the spinous layer but not in the basal layer, normal-appearing desmosomes, and prominent keratin filaments within basal keratinocytes. Thickness of cornified envelopes was reduced, lamellar lipid bilayers were disturbed, lamellar body secretion occurred prematurely and processing of secreted lamellar body contents was delayed. These barrier abnormalities were reminiscent of (albeit less severe than in) Netherton syndrome, which results from a deficiency of the serine protease inhibitor LEKTI. This work describes ultrastructural findings with evidence of epidermal barrier abnormalities in AREI.

Rapid and easy diagnosis of Netherton syndrome with dermoscopy.

BACKGROUND: Netherton syndrome is a rare autosomal recessive disease demonstrating ichthyosis linearis circumflexa, atopic findings, and hair shaft anomalies. Trichorrhexis invaginata is the pathognomonic hair shaft anomaly seen in this syndrome. OBJECTIVE: In recent years, hair shaft anomalies have been described as "matchstick" and "golf tee" signs. We present a patient with Netherton syndrome diagnosed by the presence of matchstick and golf tee hairs in addition to trichorrhexis invaginata.

Netherton syndrome: skin inflammation and allergy by loss of protease inhibition.

Netherton syndrome (NS) is a rare autosomal recessive skin disease with severe skin inflammation and scaling, a specific hair shaft defect and constant allergic manifestations. NS is caused by loss-of-function mutations in SPINK5 (serine protease inhibitor of kazal type 5) encoding LEKTI-1 (lympho-epithelial kazal type related inhibitor type 5) expressed in stratified epithelia. In vitro and in vivo studies in murine models and in NS patients have cast light on the pathogenesis of the disease and shown that LEKTI deficiency results in unopposed kallikrein-related peptidase 5 (KLK5) and KLK7 activities and to the overactivity of a new epidermal protease, elastase 2 (ELA2). Two main cascades initiated by KLK5 activity have emerged. One results in desmoglein 1 degradation and desmosome cleavage leading to stratum corneum detachment. KLK5 also activates KLK7 and ELA2, which contribute to a defective skin barrier. This facilitates allergen and microbe penetration and generates danger signals leading to caspase 1 activation and the production of active interleukin-1ß. In parallel, KLK5 activates a specific cascade of allergy and inflammation by activating protease-activated receptor-2 (PAR-2) receptors. PAR-2 activation triggers the production of the major pro-Th2 cytokine TSLP (thymic stromal lymphopoietin) and several inflammatory cytokines, including tumour necrosis factor-α. Levels of thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) also contribute to allergy in a PAR-2-independent manner. Patient investigations have confirmed these abnormalities and revealed a wide spectrum of disease expression, sometimes associated with residual LEKTI expression. These results have demonstrated that the tight regulation of epidermal protease activity is essential for skin homeostasis and identified new targets for therapeutic intervention. They also provide a link with atopic dermatitis through deregulated protease activity, as recently supported by functional studies of the E420K LEKTI variant.

rAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients.

BACKGROUND: Netherton syndrome (NS, MIM 256500) is a potential live threatening autosomal-recessive skin disorder clinically characterized by the trias of congenital erythroderma, hair shaft anomalies and atopic diathesis. It is caused by mutations in the gene SPINK5 resulting in a deficiency of its processed protein named lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI controls the activity of several serine proteases in the skin that are involved in terminal differentiation. Loss of LEKTI results in protease hyperactivity, increased degradation of intercellular junctions, reduced stratum corneum adhesion and impaired skin barrier function. Today NS can only be treated symptomatically. OBJECTIVE: Does gene transfer offer a therapeutic option for NS in the future? METHODS: A recombinant adeno-associated virus type 2 vector was constructed containing the full length cDNA (rAAV2/C-SPINK5) of functional human LEKTI. Infectious virus particles were used for transfection of LEKTI-deficient-keratinocytes of NS patients in vitro. RESULTS: Gene transfer of SPINK5 in NS-keratinocytes led to a five-fold increase in mRNA expression of SPINK5 reaching almost 75% of normal value. The functionality of the expressed LEKTI was proven in a hydrolytic activity assay demonstrating that the activity of LEKTI after gene transfer increased closely to the level seen in keratinocytes of healthy individuals. CONCLUSION: The results provide first evidence that gene transfer of SPINK5 results in increased LEKTI activity in NS-keratinocytes, thus offering a rational to further pursue such a gene therapy approach for NS.

Early skin biopsy is helpful for the diagnosis and management of neonatal and infantile erythrodermas.

BACKGROUND: Erythrodermas are often life-threatening conditions in infants. Determination of the underlying cause is crucial. Microscopic changes in adult erythroderma lack specificity. OBJECTIVE: To determine if an early skin biopsy is helpful for the diagnosis of neonatal and infantile erythroderma. METHODS: Seventy-two patients admitted for erythroderma in the first year of life were retrospectively included. One hundred and eleven skin biopsies (12-year period) were examined by 3 pathologists blinded to the clinical diagnosis, and classified into atopic dermatitis, immunodeficiency (ID), psoriasis, Netherton syndrome (NS), ichthyosis, other. From year 2000, LEKTI antibody was performed when NS was suspected. Pathological diagnosis was then compared with clinical diagnosis. RESULTS: The final diagnosis was made in 69.3% of the cases. In 57.6%, pathological diagnosis was in accordance, and in 11.7%, it was in accordance, but other diagnosis had also been proposed. For ID, sensitivity and specificity were 58.5 and 98.5%, respectively. Before year 2000, NS was frequently misdiagnosed with psoriasis, but with the use of LEKTI antibody, sensitivity and specificity were 100%. CONCLUSION: Skin biopsy is helpful for etiologic diagnosis of early erythroderma of infancy, particularly in ID and NS, the most severe diseases. Consequently, these results justify an early systematic skin biopsy for a better and earlier management.