Dominant dystrophic epidermolysis bullosa is associated with glycolytically active GATA3+ Th2 cells which may contribute to pruritus in lesional skin.

BACKGROUND: Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by trauma-induced blisters and, in some individuals, intense pruritus. Precisely what causes itch in DDEB and optimal ways to reduce it have not been fully determined. OBJECTIVE: To characterize DDEB skin transcriptomes to identify therapeutic targets to reduce pruritus in patients. METHODS: We evaluated affected and unaffected skin biopsy samples from 6 DDEB subjects (all with the very itchy pruriginosa subtype), and 4 healthy individuals using bulk RNA-seq. Single-cell transcriptomes of affected (n=2) and unaffected (n=1) DDEB and healthy skin (n=2) were obtained. Dupilumab treatment was provided for three patients. RESULTS: The skin bulk transcriptome showed significant enrichment of Th1/2 and Th17 pathways in affected DDEB skin compared with non-lesional DDEB and healthy skin. Single-cell transcriptomics showed an association of glycolytically active GATA3+ Th2 cells in affected DDEB skin. Treatment with dupilumab in three people with DDEB led to significantly reduced VAS itch scores after 12 weeks (mean VAS=3.83) compared to pre-treatment (mean VAS=7.83). Bulk RNA-seq and qPCR showed that healthy skin and dupilumab-treated epidermolysis bullosa (EB) pruriginosa skin show very similar transcriptomic profiles, and reduced Th1/2 and Th17 pathway enrichment. CONCLUSIONS: Single-cell RNA-seq helps define an enhanced DDEB-associated Th2 profile and rationalizes drug repurposing of anti-Th2 drugs in treating DDEB pruritus.

Tofacitinib ameliorates skin inflammation in a case of severe autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous disorder manifesting aberrant skin scaling and increased transepidermal water loss (TEWL). Current treatments for ARCI are limited and sub-optimal. We studied a 27-year-old man with ARCI resulting from a homozygous missense variant in TGM1 (transglutaminase 1). RNA-sequencing of lesional skin revealed aberrant JAK-STAT signalling, providing a rationale for innovative treatment with a Janus kinase inhibitor. We prescribed oral tofacitinib (11 mg daily) for 26 weeks. Rapid improvements in erythema and fissuring manifested within the first month. Sustained reductions in 5-D itch scale and Dermatology Life Quality Index (DLQI) scores were also observed. TEWL decreased for the first 10 weeks but increased thereafter. Tofacitinib down-regulated inflammatory genes and pathways, while enhancing skin barrier markers. Moreover, TGM1 distribution was normalized although enzymatic activity remained deficient. This study suggests that oral tofacitinib may be a useful therapy to consider in patients with ARCI.

Transcriptomic responses of peripheral blood mononuclear cells to cyclosporin and etanercept in a female infant with juvenile generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a rare but severe form of psoriasis. An early onset of the diseases is correlated with mutations among IL36RN, CARD14, AP1S3, MPO and SERPINA3 genes. Systemic biological agents including anti-TNF-α, anti-IL-17, anti-IL-12/IL-23, anti-IL1R, anti-IL1ß and anti-IL-36R act as novel treatment methods for GPP. Herein we report a female infant clinically diagnosed with GPP since she was 10-month-old. Results of whole-exome sequencing (WES) and Sanger sequencing revealed a reported heterozygous IL36RN (c.115+6T>C) and another reported heterozygous SERPINA3 frame-shifting variant (c.1247_1248del). Initial cyclosporin treatment for the patient led to a partial remission of the symptoms. However, the patient reached nearly total remission of pustules and erythema after anti-TNF-α inhibitor etanercept treatment. Results of further RNA sequencing (RNA-seq) done on peripheral blood mononuclear cells correlated with the clinical responses, showing that cyclosporin suppressed a portion of the neutrophil-related genes, while most genes associated with neutrophil activation, neutrophil-mediated immunity and degranulation were downregulated by the subsequent etanercept treatment. We report this case to demonstrate WES and RNA-seq in combination could come in handy in reaching a precise diagnosis and in evaluating or even predicting the molecular alterations underlying clinical treatment effectiveness.

TIMP3/Wnt axis regulates gliosis of Müller glia.

BACKGROUND: Previous studies have confirmed the expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in Müller glia (MG). However, the role of TIMP3 in MG remains unknown. METHODS: A mouse model of laser-induced retinal damage and gliosis was generated using wild-type C57BL/6 mice. TIMP3 and associated proteins were detected using Western blotting and immunofluorescence microscopy. RNA sequencing (GSE132140) of mouse laser-induced gliosis was utilized for pathway analysis. TIMP3 overexpression was induced in human MG. Human vitreous samples were obtained from patients with proliferative diabetic retinopathy (PDR) and healthy controls for protein analysis. RESULTS: TIMP3 levels increased in mouse eyes after laser damage. Morphology and spatial location of TIMP3 indicated its presence in MG. TIMP3-overexpressing MG showed increased cellular proliferation, migration, and cell nuclei size, suggesting TIMP3-induced gliosis for retinal repair. Glial fibrillary acidic protein (GFAP) and vimentin levels were elevated in TIMP3-overexpressing MG and laser-damaged mouse retinas. RNA sequencing and Western blotting suggested a role for ß-catenin in mediating TIMP3 effects on the retina. Human vitreous samples from patients with PDR showed a positive correlation between TIMP3 and GFAP levels, both of which were elevated in patients with PDR. CONCLUSIONS: TIMP3 is associated with MG gliosis to enhance the repair ability of damaged retinas and is mediated by the canonical Wnt/ß-catenin. Changes in TIMP3 could potentially be used to control gliosis in a range of retinal diseases However, given the multifaceted nature of TIMP3, care must be taken when developing treatments that aim solely to boost the function of TIMP3. FUNDING: National Cheng Kung University Hospital, Taiwan (NCKUH-10604009 and NCKUH-11202007); the Ministry of Science and Technology (MOST 110-2314-B-006-086-MY3).

Adipose transcriptome in the scalp of androgenetic alopecia.

Previous studies have shown how adipocytes can modulate the activity of hair follicle stem cells. However, the role of adipocytes in the pathogenesis of androgenetic alopecia (AGA) remains unknown. We aimed to determine signaling pathways related to the adipose tissue changes in the human scalp with AGA through RNA-seq analysis. RNA was isolated from the adipose tissues derived from the bald (frontal) and normal (occipital) scalps of male patients with AGA (n = 4). The pooled RNA extracts from these samples were subjected to RNA sequencing, followed by differential gene expression and pathway analysis. Our gene expression analysis identified 1,060 differentially expressed genes, including 522 upregulated and 538 downregulated genes in the bald AGA scalp. Biological pathways pertaining to either adipose tissue metabolism or the hair cycle were generated in our pathway analysis. Downregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway was noted to be significant in the bald scalp. Expression of adipogenic markers (e.g., PPARG, FABP4, PLN1, and ADIPOQ) was also decreased in the bald site. These findings imply that adipogenesis becomes downregulated in AGA, specifically within the bald scalp adipose. Our results lead to the hypothesis that PPARγ-mediated adipogenesis in the scalp adipose, via crosstalk with signaling pathways involved in hair cycling, might play a role in AGA.

Genetic Diagnosis of Rubinstein-Taybi Syndrome With Multiplex Ligation-Dependent Probe Amplification (MLPA) and Whole-Exome Sequencing (WES): Case Series With a Novel CREBBP Variant.

Rubinstein-Taybi Syndrome (RSTS) is a rare congenital disease with distinctive facial features, broadening of the thumbs and halluces, and developmental delay. RSTS is caused by de novo genetic alterations in CREBBP and the homologous EP300 genes. In this study, we established a genetic diagnostic protocol by integrating multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES). Five patients clinically diagnosed with RSTS were enrolled for genetic testing. Germline DNA was extracted from the peripheral blood of the patients and their families. One patient (case 1) was identified as harboring a large heterozygous deletion in the 16p13.3 region, spanning the CREBBP gene. Three patients (Cases 2-4) harbored different CREBBP variants (c.2608C>T:p.Gln870Ter,c.4404_4405del:p.Thr1468fs,c.3649C>T:p.Gln1217Ter). No causative variants were identified for the fifth RSTS patient (case 5). Here, we propose a molecular diagnostic protocol that identified causative genetic alterations in 4/5 of the patients, yielding a molecular diagnostic rate of 80%. Given the rarity of RSTS, more research is needed to explore its pathogenesis and mechanism.