[A case of neonatal Netherton syndrome]. / 新生儿Netherton综合征1例.

A male infant, aged 6 days, was admitted to the hospital due to respiratory distress and systemic desquamative rash after birth. The infant presented with erythema and desquamative rash, respiratory failure, recurrent infections, chronic diarrhea, hypernatremic dehydration, and growth retardation. Comprehensive treatment, including anti-infection therapy, intravenous immunoglobulin administration, and skin care, resulted in improvement of the rash, but recurrent infections persisted. Second-generation sequencing revealed a homozygous mutation in the SPINK5 gene, consistent with the pathogenic variation of Netherton syndrome. The family opted for palliative care, and the infant died at the age of 2 months after discharge. This report documents a case of Netherton syndrome caused by the SPINK5 gene mutation in the neonatal period, and highlights multidisciplinary diagnosis and therapy for this condition.

Genome-wide association study identifies kallikrein 5 in type 2 inflammation-low asthma.

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.

Cocktails of KLK5 Protease Inhibitors and Anti-TNFα Therapeutics: an Effective Treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare, severe type of ichthyosis, often lethal in neonates, for which there is no therapy. Spink5-/- mice recapitulate major NS hallmarks and die homogeneously within 5 h from birth due to severe epidermal barrier defect leading to dehydration. Spink5-/-Klk5-/- mice survive neonatal lethality, indicating that KLK5 could be a drug target for NS. Nevertheless, after a week, these mice developed epidermal inflammation and signs of barrier defect leading to lethality. Here we tested whether anti-TNFα strategy in combination with anti-KLK5 could provide a long-term effective therapy for NS. Deletion of Tnfa in Spink5-/- suppressed the inflammatory phenotype but did not rescue neonatal lethality of Spink5-/- indicating that anti-TNFα therapy alone would not be sufficient to treat NS. Interestingly, in Spink5-/-Klk5-/-Tnfa-/- mice, NS features were rescued, and mice lived normally for 16-18 months. For the first time, evidence is provided that a combination of anti-TNFα and anti-KLK5 therapeutics represents an effective therapeutic strategy for NS. Notably, anti-TNFα factors are marketed and used widely, while LMW KLK5 inhibitors are being developed.

STAT3 maintains skin barrier integrity by modulating SPINK5 and KLK5 expression in keratinocytes.

Skin barrier dysfunction induces skin inflammation. Signal transducer and activator of transcription 3 (STAT3) is known to be involved in Th17-mediated immune responses and barrier integrity in the cornea and intestine; however, its role in the skin barrier remains largely unknown. In this study, we elucidated the potential role of STAT3 in the skin barrier and its effect on kallikrein-related peptidase 5 (KLK5) and serine protease inhibitor Kazal-type 5 (SPINK5) expression using a mouse model with keratinocyte-specific ablation of STAT3. Keratinocyte-specific loss of STAT3 induced a cutaneous inflammatory phenotype with pruritus and intense scratching behaviour in mice. Transcriptomic analysis revealed that the genes associated with impaired skin barrier function, including KLK5, were upregulated. The effect of STAT3 on KLK5 expression in keratinocytes was not only substantiated by the increase in KLK5 expression following treatment with STAT3 siRNA but also by its decreased expression following STAT3 overexpression. Overexpression and IL-17A-mediated stimulation of STAT3 increased the expression of SPINK5, which was blocked by STAT3 siRNA. These results suggest that the expression of SPINK5 and KLK5 in keratinocytes could be dependent on STAT3 and that STAT3 might play an essential role in the maintenance of skin barrier homeostasis.

Metformin inhibits melanoma cell metastasis by suppressing the miR-5100/SPINK5/STAT3 axis.

Melanoma is the most lethal skin cancer characterized by its high metastatic potential. It is urgent to find novel therapy strategies to overcome this feature. Metformin has been confirmed to suppress invasion and migration of various types of cancer. However, additional mechanisms underlying the antimetastatic effect of metformin on melanoma require further investigation. Here, we performed microarray analysis and uncovered an altered mRNA and miRNA expression profile between melanoma and nevus. Luciferase reporter assay confirmed that miR-5100 targets SPINK5 to activate STAT3 phosphorylation. Migration and wound healing assays showed that the miR-5100/SPINK5/STAT3 axis promotes melanoma cell metastasis; the mechanism was proven by initiation of epithelial-mesenchymal transition. Co-immunoprecipitation (Co-IP) further confirmed an indirect interaction between SPINK5 and STAT3. Furthermore, metformin dramatically inhibited miR-5100/SPINK5/STAT3 pathway, and decreased B16-F10 cell metastasis to lung in C57 mouse module. Intriguingly, pretreatment of metformin before melanoma cell injection improved this effect further. These findings exposed the underlying mechanisms of action of metformin and update the use of this drug to prevent metastasis in melanoma.

Lobelia chinensis Extract and Its Active Compound, Diosmetin, Improve Atopic Dermatitis by Reinforcing Skin Barrier Function through SPINK5/LEKTI Regulation.

The skin acts as a mechanical barrier that protects the body from the exterior environment, and skin barrier function is attributed to the stratum corneum (SC), which is composed of keratinocytes and skin lipids. Skin barrier homeostasis is maintained by a delicate balance between the differentiation and exfoliation of keratinocytes, and keratinocyte desquamation is regulated by members of the serine protease kalikrein (KLK) family and their endogenous inhibitor SPINK5/LEKTI (serine protease inhibitor Kazal type 5/lympho-epithelial Kazal-type-related inhibitor). Furthermore, SPINK5/LEKTI deficiency is involved in impaired skin barrier function caused by KLK over-activation. We sought to determine whether increased SPINK5/LEKTI expression ameliorates atopic dermatitis (AD) by strengthening skin barrier function using the ethanol extract of Lobelia chinensis (LCE) and its active compound, diosmetin, by treating human keratinocytes with UVB and using a DNCB-induced murine model of atopic dermatitis. LCE or diosmetin dose-dependently increased the transcriptional activation of SPINK5 promoter and prevented DNCB-induced skin barrier damage by modulating events downstream of SPINK5, that is, KLK, PAR2 (protease activated receptor 2), and TSLP (thymic stromal lymphopoietin). LCE or diosmetin normalized immune response in DNCB treated SKH-1 hairless mice as determined by reductions in serum immunoglobulin E and interleukin-4 levels and numbers of lesion-infiltrating mast cells. Our results suggest that LCE and diosmetin are good candidates for the treatment of skin barrier-disrupting diseases such as Netherton syndrome or AD, and that they do so by regulating SPINK5/LEKTI.

Comel-Netherton syndrome: A local skin barrier defect in the absence of an underlying systemic immunodeficiency.

BACKGROUND: Comel-Netherton syndrome (NS) is a rare autosomal disease, characterized by severe skin disease, hair shaft defects, atopic diathesis, and increased susceptibility for skin infections. Since patients with NS suffer from recurrent infections, it has been hypothesized that an underlying immunodeficiency attributes to this. Here, we studied clinical and immunological characteristics of the cohort of NS patients in the Netherlands in order to identify whether potential immunodeficiencies result in the increased risk of infectious complications. METHODS: Phenotypes were scored for severity of skin condition, specific hair shaft defects, atopy, and recurrent infections. Patients' blood samples were collected for quantification of serum immunoglobulin (Ig) levels, specific antibodies against Streptococcus pneumoniae, and allergen-specific IgE, as well as detailed immunophenotyping of blood leukocyte and lymphocyte subsets by flow cytometry. RESULTS: A total of 14 patients were included with age range 3-46 years and varying degrees of skin involvement. All patients presented with atopic symptoms (food allergy, n = 13; hay fever, n = 10; asthma, n = 7). Recurrent skin infections were common, particularly in childhood (n = 12). Low levels of specific antibodies against S pneumoniae were found in 10 of 11 evaluated patients. Detailed immunological analysis was performed on 9 adult patients. Absolute numbers of lymphocyte subsets and serum immunoglobulin levels were all within normal ranges. CONCLUSION: Multidisciplinary evaluation of our national cohort showed no evidence for a severe, clinically relevant systemic immunodeficiency. Therefore, we conclude that in Dutch NS patients the increased risk of infections most likely results from the skin barrier disruption and that increased allergen penetration predisposes to allergic sensitization.

Multifaceted Analyses of Epidermal Serine Protease Activity in Patients with Atopic Dermatitis.

The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals (n = 46) and AD patients (n = 63) by tape stripping and then measuring the trypsin- and chymotrypsin-like serine protease activity. We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC's serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.

A New Splice-site Mutation of SPINK5 Gene in the Netherton Syndrome with Different Clinical Features: A Case Report.

Netherton syndrome (NS) is a rare genodermatosis characterized by the triad of ichthyosiform erythroderma, hair shaft abnormality and an atopic diathesis. We report a case of a 20-year-old male patient presented with pruritus, decreased sweat secretion and generalized erythema on his body. Netherton syndrome is caused by mutations in the SPINK5 gene that is a crucial role for epidermal barrier function in the skin. Different clinical and phenotypical features can occur based on various LEKTI-domains mutations. Diagnosis is made by the atopic story, hair shaft abnormality, cutaneous lesions and identification of the SPINK5 gene mutation. In our patient, we detected a new splice site mutation in the SPINK5 gene and pili annulati as hair abnormality. Affected patients are usually misdiagnosed because of cutaneous lesions such as atopic dermatitis. Therefore, each clinical finding should be evaluated together. We aimed to present a case with a new SPINK5 gene mutation and different clinical features in NS.

Hyper IgE syndromes: clinical and molecular characteristics.

Hyper IgE syndromes comprise a group of rare primary immunodeficiency disorders characterized by a triad of atopic dermatitis, recurrent skin and lung infections along with elevated IgE levels. Job syndrome or autosomal dominant hyper IgE syndrome because of heterozygous loss-of-function mutations with dominant negative effect in signal transducer and activator of transcription-3 is the prototype of these disorders. However, several other genetically characterized immunodeficiency disorders have been identified over the past decade and joined the umbrella of hyper IgE syndromes including autosomal recessive mutations in the DOCK8, ZNF431 and PGM3 genes and heterozygous mutations with dominant negative effect in the CARD11 gene. Moreover, a number of phenotypically distinct immunodeficiency disorders can mimic hyper IgE syndromes, adding to the diagnostic challenge. Herein, we will concisely review these disorders, their molecular bases, highlighting key distinguishing clinical and laboratory findings and therapeutic options.

Structure guided drug design to develop kallikrein 5 inhibitors to treat Netherton syndrome.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.

SPINK5 is associated with early-onset and CHI3L1 with late-onset atopic dermatitis.

We have recently showed that filaggrin (FLG) mutations are associated only with early-onset of AD, but not with late-onset of AD. Consequently, other susceptibility genes should receive attention, especially in patients with late-onset of AD. Our aim was to assess the associations between development of AD and the polymorphisms rs2303067 in SPINK5 and rs490928 in CHI3L1. A study population of 241 AD patients and 164 healthy controls was genotyped for two polymorphisms (rs2303067 in SPINK5 and rs490928 in CHI3L1). Rs2303067 in SPINK5 was significantly associated with early-onset AD (≤8 years: p = .003; OR = 2.57) and was characterized by the need for hospitalization (p = .006; OR = 2.76), prolonged duration (≥10 years; p = .008; OR = 2.32) and more body parts affected (p = .015; OR = 2.01). In contrast, rs490928 in CHI3L1 was associated with late-onset AD (>8 years: p = .048; OR = 1.65) and was characterized by no need for hospitalization (p = .049; OR = 1.59), shorter duration (<10 years; p = .017; OR = 1.94) and fewer body parts affected (p = .049; OR = 1.75). Our results confirmed that different AD phenotypes, specifically early- and late-onset AD, have different genetic backgrounds. Early-onset AD was associated with rs2303067 in SPINK5, which is involved in skin barrier functioning, and late-onset was associated with rs4950928 in CHI3L1, which is involved in the immune response. Future studies should examine the early- versus late-onset subgrouping more closely.

Is c.1431-12G>A A common European mutation of SPINK5? report of a patient with Netherton Syndrome.

Netherton Syndrome (NS) is a very rare genetic skin disease resulting from defects in the SPINK5 gene (encoding the protease inhibitor lympho-epithelial Kazal type inhibitor 1, LEKTI1). In this report, we provide a detailed clinical description of a Polish patient with two SPINK5 mutations, the novel c.1816_1820+21delinsCT and possibly recurrent c.1431-12G>A. A detailed pathogenesis of Netherton Syndrome, on the basis of literature review, is discussed in the view of current knowledge about the LEKT1 molecular processing and activity.

Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2ß splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2ß-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes.

SPINK5 and ADRB2 haplotypes are risk factors for asthma in Mexican pediatric patients.

BACKGROUND: Asthma is one of the most common respiratory diseases worldwide, and the complexity of its etiology has been widely documented. Chromosome 5q31-33 is one of the main loci implicated in asthma and asthma-related traits. IL13, CD14 and ADRB2, which are located in this risk locus, are among the genes most strongly associated with asthma susceptibility. OBJECTIVES: This study evaluated whether single-nucleotide polymorphisms or haplotypes at 5q31-33 conferred risk for asthma in Mexican-Mestizo pediatric patients. METHODS: We performed a case-controlled study including 851 individuals, 421 of them affected with childhood-onset asthma and 430 ethnically matched unaffected subjects. We used the TaqMan Allelic Discrimination Assay to genotype 20 single-nucleotide polymorphisms within IL5, RAD50, IL13, IL4, CD14, SPINK5, HTR4, ADRB2 and IL12B. RESULTS: Although no association was detected for any risk allele, three SPINK5 haplotypes (GGCT: p = 6 × 10(-6); AATC: p = 0.0001; AGTT: p = 0.0001) and five ADRB2 haplotypes (AGGACC: p = 0.0014; AGGAAG: p = 0.0002; TGAGAG: p = 0.0001; AGGAAC: p = 0.0002; AAGGAG: p = 0.003) were associated with asthma. Notably, the AGTT SPINK5 haplotype exhibited a male gender-dependent association (p = 7.6 × 10(-5)). CONCLUSION: Our results suggest that SPINK5 and ADRB2 haplotypes might play a role in the susceptibility to childhood-onset asthma.