Comparative analyses of Netherton syndrome patients and Spink5 conditional knock-out mice uncover disease-relevant pathways.

Netherton syndrome (NS) is a rare skin disease caused by loss-of-function mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) gene. Disease severity and the lack of efficacious treatments call for a better understanding of NS mechanisms. Here we describe a novel and viable, Spink5 conditional knock-out (cKO) mouse model, allowing to study NS progression. By combining transcriptomics and proteomics, we determine a disease molecular profile common to mouse models and NS patients. Spink5 cKO mice and NS patients share skin barrier and inflammation signatures defined by up-regulation and increased activity of proteases, IL-17, IL-36, and IL-20 family cytokine signaling. Systemic inflammation in Spink5 cKO mice correlates with disease severity and is associated with thymic atrophy and enlargement of lymph nodes and spleen. This systemic inflammation phenotype is marked by neutrophils and IL-17/IL-22 signaling, does not involve primary T cell immunodeficiency and is independent of bacterial infection. By comparing skin transcriptomes and proteomes, we uncover several putative substrates of tissue kallikrein-related proteases (KLKs), demonstrating that KLKs can proteolytically regulate IL-36 pro-inflammatory cytokines. Our study thus provides a conserved molecular framework for NS and reveals a KLK/IL-36 signaling axis, adding new insights into the disease mechanisms and therapeutic targets.

[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.

Netherton Syndrome Caused by Heterozygous Frameshift Mutation Combined with Homozygous c.1258A>G Polymorphism in SPINK5 Gene.

Netherton syndrome (NS) is a rare autosomal recessive disorder caused by SPINK5 mutations, resulting in a deficiency in its processed protein LEKTI. It is clinically characterized by the triad of congenital ichthyosis, atopic diathesis, and hair shaft abnormalities. The SPINK5 (NM_006846.4): c.1258A>G polymorphism (rs2303067) shows a significant association with atopy and atopic dermatitis (AD), which share several clinical features with NS. We describe an NS patient, initially misdiagnosed with severe AD, who carried the heterozygous frameshift (null) mutation (NM_006846.4): c.957_960dup combined with homozygous rs2303067 in the SPINK5 gene. Histopathological examination confirmed the diagnosis, whereas an immunohistochemical study showed normal epidermal expression of LEKTI, despite the genetic findings. Our results corroborate the hypothesis that haploinsufficiency of SPINK5, in the presence of a SPINK5 null heterozygous mutation in combination with homozygous SPINK5 rs2303067 polymorphism, can be causative of an NS phenotype, impairing the function of LEKTI despite its normal expression. Due to the clinical overlap between NS and AD, we suggest performing SPINK5 genetic testing to search for the SPINK5 (NM_006846.4): c.1258A>G polymorphism (rs2303067) and ensure a correct diagnosis, mainly in doubtful cases.

Clinical and genetic characterization of Netherton syndrome due to SPINK5 founder variant in Latvian population.

OBJECTIVE: Netherton syndrome (NS) (OMIM:256500) is a very rare autosomal recessive multisystem disorder mostly affecting ectodermal derivatives (skin and hair) and immune system. It is caused by biallelic loss-of-function variants in the SPINK5 gene, encoding the protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI). MATERIAL, METHODS AND RESULTS: Here, we describe NS clinical and genetic features of homogenous patient group: 9 individuals from 7 families with similar ethnic background and who have the same SPINK5 variant (NM_006846.4: c.1048C > T, p.(Arg350*)) in homozygous or compound heterozygous states, suggesting that it is a common founder variant in Latvian population. Indeed, we were able to show that the variant is common in general Latvian population, and it shares the same haplotype among the NS individual. It is estimated that the variant arose >1000 years ago. Clinically, all nine patients exhibited typical NS skin changes (scaly erythroderma, ichthyosis linearis circumflexa, itchy skin), except for one patient who has a different skin manifestation-epidermodysplasia. Additionally, we show that developmental delay, previously underrecognized in NS, is a common feature among these patients. CONCLUSIONS: This study shows that the phenotype of NS individuals with the same genotype is highly homogeneous.

Netherton syndrome in a Bulgarian patient : Presentation of a case and an update of therapeutic options.

Comel-Netherton syndrome, or Netherton syndrome (NS), is a rare chronic genetic skin condition affecting the daily life of patients, which often results in poorly developed social skills and anxiety. Genetic predisposition plays a key role alongside the clinical findings, and clinicians must be aware of it as it can mimic other well-known skin conditions. Diagnosis is challenging both clinically and histologically. Clinically, it can mimic a severe form of atopic dermatitis, psoriasiform dermatitis overlapping with atopic dermatitis, or erythrokeratodermia variabilis. The difficulties in making histological diagnosis are similar, and it is often necessary to take several biopsies in order to clarify the diagnosis. Although retinoids are used for both psoriasis, erythrokeratodermia variabilis, and other congenital forms of keratodermia, the recommended treatment doses are different. This often results in poor treatment outcome. We present a 16-year-old patient previously diagnosed as erythrokeratodermia variabilis and treated with little to no improvement. Systemic therapy with acitretin 10 mg daily, local pimecrolimus 1%, emollients, and bilastine 20 mg once daily was initiated. Due to the limited application of retinoids and the difficulties in achieving permanent remission, modern medicine is faced with the challenge of seeking innovative therapeutic solutions. New hopes are placed on targeted or anti-cytokine therapy, based on inhibiting the inflammatory component of the disease. This article is mainly focused on innovative therapeutic options, including modern medications such as dupilumab, infliximab, secukinumab, anakinra, omalizumab, and others.

Ustekinumab therapy for Netherton syndrome.

Netherton syndrome (NS) is a rare disorder of cornification associated with high morbidity. It is caused by bi-allelic mutations in SPINK5 encoding the serine protease inhibitor LEKTI. Previous studies have shown Th17 skewing with IL-23 upregulation in NS, raising the possibility that targeting these inflammatory pathways may alleviate disease manifestations. We ascertained the therapeutic efficacy of six doses of ustekinumab administered to three patients with NS over a period of 13 months using the Ichthyosis Area and Severity Index (IASI), the Dermatology Life Quality Index (DLQI), a visual analogue scale (VAS) for itch and the peak-pruritus numeric rating scale (PP-NRS). Histopathology analysis including CD3, CD4, CD8 and interleukin 17 (IL-17) immunostaining, was performed at baseline and 4 weeks following the last ustekinumab dose. Total IASI scores were reduced by 28% in two patients at week 16 with sustained response by week 56. No consistent improvement in DLQI, VAS for itch and PP-NRS scores was observed. The inflammatory infiltrate and the degree of acanthosis were slightly reduced at week 56 as compared to baseline. No significant change in immunostaining of the various inflammatory markers was observed at week 56. In conclusion, this case series did not demonstrate a significant therapeutic effect of ustekinumab in NS.

Dupilumab improves clinical symptoms in children with Netherton syndrome by suppressing Th2-mediated inflammation.

Background: Netherton syndrome is a rare, life-threatening autosomal recessive genetic disorder with no effective treatment yet. Skin barrier dysfunction caused by SPINK5 gene mutations is a hallmark of the disease. Antigen penetration through the defective skin and nonspecific inflammation provide a pro-T helper 2 (Th2) immune microenvironment in the disease. Therefore, Th2 cytokines are considered to be candidate therapeutic targets. Objective: To evaluate the clinical responses of patients with Netherton syndrome to dupilumab, an IL-4Rα antagonist, and identify changes in the Th1/2/17 pathway activity, skin barrier defect protein LEKTI expression after treatment. Methods: Four children with severe Netherton syndrome (aged 2 y to 4 y and 6 m) who were treated with dupilumab from January to June 2022 were evaluated at baseline, and at 4, 8, 12, 16, and 20 weeks after the start of dupilumab administration. Treatment response was assessed using the Eczema Area and Severity Index (EASI), the Numerical Rating Scale (NRS), the Dermatology Life Quality Index (CDLQI), and the Dermatitis Family Impact-questionnaire (DFI). Blood eosinophil counts, serum IgE levels and inflammatory cytokines were measured. The immunotyping of Th1/2/17 cells was performed by flow cytometry and cytokine expressions in T cell subsets were analyzed by single-cell RNA sequencing. In addition, expression of the LEKTI in skin lesions was evaluated by immunohistochemical analysis. Results: All four patients experienced clinical improvement, with significantly reduced EASI scores (by 75.0-83.9%) and NRS (by 87.5-90.0%) from baseline to 20 weeks of treatment. Improved quality of life scores were also seen for all patients, as measured by CDLQI and DFI. Serum IgE levels also fell by 75.6-86.9%. The serum Th2 cytokines IL-4, IL-5 and IL-13 were found at low level, with no significant changes during the treatment. However, Th2 cytokines expressed by T cells, especially IL-4, decreased at single-cell level after treatment (P = 0.029). The baseline percentage of Th2 cells (among total CD3+CD4+ T cells) was significantly higher in patients than that in healthy controls (HC) (P < 0.0001); this percentage fell from 8.25% ± 0.75% to 4.02% ± 0.62% after 20 weeks dupilumab treatment. There was no noticeable change in LEKTI protein expression in skin lesions pre- and post-treatment. Two patients reported mild ocular adverse effects, but there were no severe adverse events. Conclusion: Dupilumab may be an effective and safe treatment option in a subset of pediatric patients with Netherton syndrome, especially in improving itch and the quality of life. These effects were achieved in part by suppression of the Th2-mediated inflammation.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Establishment of a mouse model of Netherton syndrome based on CRISPR/Cas9 technology

Background: Netherton syndrome is a rare but severe autosomal recessive disorder with dominant impaired skin barrier function, caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, which encodes LEKTI (lymphoepithelial Kazal-type-related inhibitor). Objectives: To establish a murine model of Netherton syndrome based on CRISPR/Cas9 gene editing technology. Materials & Methods: Spink5-sgRNA was designed to target exon 3 of the mouse Spink5 gene. Cas9 mRNA and sgRNA were microinjected into the zygotes of C57BL/6J mice. Spink5 homozygous knockout mice were born from a heterozygous intercross, and the phenotype of these mice was compared with wild-type regarding gross morphology, histopathology and immunofluorescent detection of LEKTI. Results: Following microinjection of zygotes using the CRISPR/Cas9 system, sequencing demonstrated a 22-bp deletion at exon 3 of the mouse Spink5 gene. Histological investigation revealed complete detachment of the stratum corneum from the underlying granular layer and an absence of LEKTI in skin from Spink5 homozygous knockout mice. Conclusion: The 22-bp deleted Spink5 transgenic mouse model demonstrates the clinical phenotype and genotype of human Netherton syndrome, representing a useful tool for future gene correction and skin barrier/inflammation studies.

Netherton Syndrome.

This case report describes dry skin with marked redness of the face and hands as well as trichorrhexis invaginata.

Phage Display Selected Cyclic Peptide Inhibitors of Kallikrein-Related Peptidases 5 and 7 and Their In Vivo Delivery to the Skin.

Kallikrein-related peptidases 5 (KLK5) and 7 (KLK7) are serine proteases with homeostatic functions in the epidermis that play a critical role in Netherton syndrome (NS), a rare yet life-threatening genetic disorder that currently lacks specific treatment. Previous research suggests that controlling KLKs could lead to the development of NS therapies, but existing synthetic inhibitors have limitations. Herein, we used phage display to screen libraries comprising more than 100 billion different cyclic peptides and found selective, high-affinity inhibitors of KLK5 (Ki = 2.2 ± 0.1 nM) and KLK7 (Ki = 16 ± 4 nM). By eliminating protease-prone sites and conjugating the inhibitors to an albumin-binding peptide, we enhanced the inhibitor stability and prolonged the elimination half-life to around 5 h in mice. In tissue sections taken from mice, a fluorescently labeled peptide was detected in the epidermis, suggesting that the inhibitors can reach the KLKs upon systemic delivery and should be suited to control deregulated protease activity in NS.

Outcomes of Systemic Treatment in Children and Adults With Netherton Syndrome: A Systematic Review.

Background: Comèl-Netherton syndrome (NS) is a rare disease caused by pathogenic variants in the SPINK5 gene, leading to severe skin barrier impairment and proinflammatory upregulation. Given the severity of the disease, treatment of NS is challenging. Current treatment regimens are mainly topical and supportive. Although novel systemic treatment options for NS have been suggested in recent literature, little is known about their outcomes. Objective: to provide an overview of systemic treatment options and their outcomes in adults and children with NS. Methods: Embase, MEDLINE, Web of Science, Cochrane Central Register of Controlled Trials, and Google Scholar were searched up to July 22, 2021. Empirical studies published in English language mentioning systemic treatment in NS were enrolled. Studies that did not define a treatment period or report at least one outcome were excluded. Methodological quality was evaluated by the Joanna Briggs Institute critical appraisal checklist for case reports or case series. Overall quality of evidence of the primary outcome, skin, was assessed by the GRADE approach. Results: 36 case series and case reports were included. The effects of 15 systemic therapies were described in 48 patients, of which 27 were children. Therapies included retinoids, prednisolone, cyclosporine, immunoglobulins, and biologicals. In retinoids both worsening (4/15 cases) and improvement (6/15 cases) of the skin was observed. Use of prednisolone and cyclosporine was only reported in one patient. Immunoglobulins (13/15 cases) and biologicals (18/21 cases) showed improvement of the skin. Certainty of evidence was rated as very low. Conclusion: NS is a rare disease, which is reflected in the scarce literature on systemic treatment outcomes in children and adults with NS. Studies showed large heterogeneity in outcome measures. Adverse events were scarcely reported. Long-term outcomes were reported in a minority of cases. Nonetheless, a general beneficial effect of systemic treatment was found. Immunoglobulins and biologicals showed the most promising results and should be further explored. Future research should focus on determining a core outcome set and measurement instruments for NS to improve quality of research. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=217933, PROSPERO (ID: 217933).

A novel pathogenic variant in the corneodesmosin gene causing generalized inflammatory peeling skin syndrome with marked eosinophilia and trichorrhexis invaginata.

Generalized inflammatory peeling skin syndrome (PSS) is a rare autosomal recessive genodermatosis caused by loss-of-function disease-causing variants of the corneodesmosin gene (CDSN), resulting in excessive shedding of the superficial layers of the epidermis. We describe a case of generalized inflammatory PSS in an infant, presenting at day two of life with ichthyosiform erythroderma and superficial peeling of the skin. Hair microscopy showed trichorrhexis invaginata. Normal amounts of skin LEKT1, a product of SPINK5 on immunohistochemical staining excluded a diagnosis of Netherton syndrome. Genetic analysis revealed a homozygous novel complete CDSN deletion, estimated 4.6 kb in size, supporting the diagnosis of generalized inflammatory PSS.

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.

Netherton Syndrome in a Mother and Her Two Children.

Netherton syndrome (NS) is a rare autosomal recessive condition caused by mutations in the serine peptidase inhibitor, Kazal type 5 (SPINK5) gene which encodes for a serine protease inhibitor, lymphoepithelial Kazal-typerelated inhibitor (LEKT1). NS is characterized by a triad of ichthyosiform erythroderma, trichorrhexis invaginata, and atopic diathesis with elevated IgE levels. The syndrome typically presents in infancy, where life-threatening complications are frequent, and evolves into a less severe condition with milder clinical symptoms in adulthood. This case report details the clinical history and genetic testing of a mother and two children with clinically symptomatic and genetically proven NS.

Successful treatment of Netherton syndrome with dupilumab: A case report and review of the literature.

Netherton syndrome (NS) is a rare autosomal recessive genetic disease caused by SPINK5 gene mutation without specific effective therapies available. We report a case of NS confirmed by whole exome sequencing of DNA using peripheral blood, and Sanger sequencing found two new mutations associated with her clinical presentation located at SPINK5 gene c.1220+5G>A from her father and c.1870delA from her mother. The patient was treated with dupilumab (600 mg at week 0, then 300 mg every 2 weeks, s.c.). The clinical manifestation and dermoscopic images of the patient's hair showed remarkable improvement after dupilumab treatment with no adverse effects. We also reviewed previous reports to learn more about the therapeutic effect and adverse reactions of NS treated with dupilumab.

Netherton syndrome subtypes share IL-17/IL-36 signature with distinct IFN-α and allergic responses.

BACKGROUND: Netherton syndrome (NS) is a rare recessive skin disorder caused by loss-of-function mutations in SPINK5 encoding the protease inhibitor LEKTI (lymphoepithelial Kazal-type-related inhibitor). NS patients experience severe skin barrier defects, display inflammatory skin lesions, and have superficial scaling with atopic manifestations. They present with typical ichthyosis linearis circumflexa (NS-ILC) or scaly erythroderma (NS-SE). OBJECTIVE: We used a combination of several molecular profiling methods to comprehensively characterize the skin, immune cells, and allergic phenotypes of NS-ILC and NS-SE patients. METHODS: We studied a cohort of 13 patients comprising 9 NS-ILC and 4 NS-SE. RESULTS: Integrated multiomics revealed abnormal epidermal proliferation and differentiation and IL-17/IL-36 signatures in lesion skin and in blood in both NS endotypes. Although the molecular profiles of NS-ILC and NS-SE lesion skin were very similar, nonlesion skin of each disease subtype displayed distinctive molecular features. Nonlesion and lesion NS-SE epidermis showed activation of the type I IFN signaling pathway, while lesion NS-ILC skin differed from nonlesion NS-ILC skin by increased complement activation and neutrophil infiltration. Serum cytokine profiling and immunophenotyping of circulating lymphocytes showed a TH2-driven allergic response in NS-ILC, whereas NS-SE patients displayed mainly a TH9 axis with increased CCL22/MDC and CCL17/TARC serum levels. CONCLUSIONS: This study confirms IL-17/IL-36 as the predominant signaling axes in both NS endotypes and unveils molecular features distinguishing NS-ILC and NS-SE. These results identify new therapeutic targets and could pave the way for precision medicine of NS.

A Novel SPINK5 Gene Mutation Associated with Netherton Syndrome in an Omani Patient.

Netherton syndrome (NS) is an autosomal recessive primary immunodeficiency. It is characterised by substantial skin barrier defects and is often misdiagnosed as severe atopic dermatitis or hyper-immunoglobulin E syndrome. Although more than 80 NS-associated pathogenic mutations in the serine peptidase inhibitor kazal type 5 (SPINK5) gene have been reported worldwide, only one has been reported in the Arab population to date. We report the case of a novel association between the c.1887+1G>A mutation in the SPINK5 gene and NS in an Omani-Arab patient born in 2014 who was managed at a paediatric immunology clinic in Muscat, Oman. Accurate genetic diagnosis facilitated tailored clinical management of the index patient and enabled the provision of genetic counselling and offering of future reproductive options to the individuals related to the index patient.