Homozygous variant in TKFC abolishing triokinase activities is associated with isolated immunodeficiency.

BACKGROUND: Triokinase and FMN cyclase (TKFC) is a bifunctional enzyme involved in fructose metabolism. Triokinase catalyses the phosphorylation of fructose-derived glyceraldehyde (GA) and exogenous dihydroxyacetone (DHA), while FMN cyclase generates cyclic FMN. TKFC regulates the antiviral immune response by interacting with IFIH1 (MDA5). Previously reported pathogenic variants in TKFC are associated with either a multisystemic disease or isolated hypotrichosis with loose anagen hairs. METHODS: Whole-exome sequencing identified a homozygous novel variant in TKFC (c.1624G>A; p.Gly542Arg) in an individual with a complex primary immunodeficiency disorder. The variant was characterised using enzymatic assays and yeast studies of mutant recombinant proteins. RESULTS: The individual presented with chronic active Epstein-Barr virus disease and multiple bacterial and viral infections. Clinical investigations revealed hypogammaglobulinaemia, near absent natural killer cells and decreased memory B cells. Enzymatic assays showed that this variant displayed defective DHA and GA kinase activity while maintaining FMN cyclase activity. An allogenic bone marrow transplantation corrected the patient's immunodeficiency. CONCLUSION: Our report suggests that TKFC may have a role in the immunological system. The pathological features associated with this variant are possibly linked with DHA/GA kinase inactivation through a yet an unknown mechanism. This report thus adds a possible new pathway of immunometabolism to explore further.

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.

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis.

Acne keloidalis is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, acne keloidalis is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent no-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including 2 notable populations: POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with an M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression as well as acne keloidalis disease activity. Spatial transcriptomics and immunofluorescence staining verified microanatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of acne keloidalis.

Spatial transcriptomics in human skin research.

Spatial transcriptomics is a revolutionary technique that enables researchers to characterise tissue architecture and localisation of gene expression. A plethora of technologies that map gene expression are currently being developed, aiming to facilitate spatially resolved, high-dimensional assessment of gene transcription in the context of human skin research. Knowing which gene is expressed by which cell and in which location within skin, facilitates understanding of skin function and dysfunction in both health and disease. In this review, we summarise the available spatial transcriptomic methods and we describe their application to a broad spectrum of dermatological diseases.

A review of genotrichoses and hair pathology associated with inherited skin diseases.

Genetic hair disorders, also known as genotrichoses, are characterized by abnormalities of hair structure, growth or differentiation, giving rise to a spectrum of phenotypes such as hypertrichosis, hypotrichosis and atrichia. These disorders may present as isolated phenotypes or be part of more complex phenotypes including abnormalities in skin or other organs. Genetic discoveries for hair disorders have been recently augmented with the advent of next-generation sequencing (NGS) technologies. We reviewed the literature and summarized disease-gene associations for inherited hair disorders, as well as genodermatoses presenting with hair abnormalities discovered by NGS technologies. We identified 28 nonsyndromic hair disorders, involving 25 individual genes and four unidentified genes. We have also discovered that approximately 30% of all the genodermatoses that were identified by NGS approaches demonstrated hair abnormalities as part of their phenotype. This review underscores the huge impact of NGS technologies in disclosing the genetics of hair disorders and the potential these discoveries provide for future translational research and new therapies.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation.

Dermatofibromas (DFs) are common, benign fibrous skin tumors that can occur at any skin site. In most cases, DFs are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial DF within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation and adhesion to extracellular matrix through enhanced ß1 integrin activation and more disorganized matrix. The enzyme PLOD2 was identified as a binding partner for RhoE, and the formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen cross-linking and activation of ß1 integrins, and depleting PLOD2 in T231M-RhoE-expressing cells reduced T231M-RhoE-mediated ß1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of DF skin samples compared with that of the controls. Our data show that loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to a disorganized extracellular matrix, contributing to DF.

Comorbidities of Keloid and Hypertrophic Scars Among Participants in UK Biobank.

Importance: Keloids and hypertrophic scars (excessive scarring) are relatively understudied disfiguring chronic skin conditions with high treatment resistance. Objective: To evaluate established comorbidities of excessive scarring in European individuals, with comparisons across ethnic groups, and to identify novel comorbidities via a phenome-wide association study (PheWAS). Design, Setting, and Participants: This multicenter cross-sectional population-based cohort study used UK Biobank (UKB) data and fitted logistic regression models for testing associations between excessive scarring and a variety of outcomes, including previously studied comorbidities and 1518 systematically defined disease categories. Additional modeling was performed within subgroups of participants defined by self-reported ethnicity (as defined in UK Biobank). Of 502 701 UKB participants, analyses were restricted to 230078 individuals with linked primary care records. Exposures: Keloid or hypertrophic scar diagnoses. Main Outcomes and Measures: Previously studied disease associations (hypertension, uterine leiomyoma, vitamin D deficiency, atopic eczema) and phenotypes defined in the PheWAS Catalog. Results: Of the 972 people with excessive scarring, there was a higher proportion of female participants compared with the 229 106 controls (65% vs 55%) and a lower proportion of White ethnicity (86% vs 95%); mean (SD) age of the total cohort was 64 (8) years. Associations were identified with hypertension and atopic eczema in models accounting for age, sex, and ethnicity, and the association with atopic eczema (odds ratio [OR], 1.68; 95% CI, 1.36-2.07; P < .001) remained statistically significant after accounting for additional potential confounders. Fully adjusted analyses within ethnic groups revealed associations with hypertension in Black participants (OR, 2.05; 95% CI, 1.13-3.72; P = .02) and with vitamin D deficiency in Asian participants (OR, 2.24; 95% CI, 1.26-3.97; P = .006). The association with uterine leiomyoma was borderline significant in Black women (OR, 1.93; 95% CI, 1.00-3.71; P = .05), whereas the association with atopic eczema was significant in White participants (OR, 1.68; 95% CI, 1.34-2.12; P < .001) and showed a similar trend in Asian (OR, 2.17; 95% CI, 1.01-4.67; P = .048) and Black participants (OR, 1.89; 95% CI, 0.83-4.28; P = .13). The PheWAS identified 110 significant associations across disease systems; of the nondermatological, musculoskeletal disease and pain symptoms were prominent. Conclusions and Relevance: This cross-sectional study validated comorbidities of excessive scarring in UKB with comprehensive coverage of health outcomes. It also documented additional phenome-wide associations that will serve as a reference for future studies to investigate common underlying pathophysiologic mechanisms.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation.

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Plasma metabolomic profiling reflects the malnourished and chronic inflammatory state in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder characterized by skin fragility, chronic inflammation, malnutrition, and fibrosis. Metabolomics is an emerging investigative field that helps elucidate disease pathophysiology and identify biomarkers. However, previous metabolomic studies in RDEB are limited. OBJECTIVE: To investigate the plasma metabolomic profiles in RDEB patients. METHODS: We recruited 10 RDEB patients and 10 age-/gender-matched healthy controls. Peripheral blood samples were collected and plasma metabolomic profiling was performed by LC-MS/MS analysis. MS data processing and compound identification were executed by MS-DIAL. Enrichment analysis was performed by MetaboAnalyst 5.0. RESULTS: Metabolomic analyses demonstrated that most amino acid levels were downregulated in RDEB patients, and the extent of insufficiency correlated with clinical severity. Several metabolites were dysregulated in RDEB, including glutamine and glutamate metabolism, tryptophan-to-kynurenine ratio, phenylalanine-to-tyrosine ratio, and succinate accumulation. LIMITATIONS: The study was limited by small case numbers and the unrepresentativeness of a single time-point blood sample. CONCLUSION: Our study demonstrated the altered metabolomic profiles in RDEB, reflecting the disease severity, the chronic inflammatory and malnourished status, while the fibrotic signatures were not evident.

The Past and Future of Rare Skin Disease Research and Therapy.

Launch of the European Society for Dermatological Research (ESDR) in 1970 coincided with genetics also entering a new era. Arriving alongside new models of DNA structure and the discovery of restriction endonucleases, the ESDR has parallel-tracked 50 years of major developments in genomics, technological innovations, and big data. Patients with rare Mendelian genetic skin diseases have witnessed the discovery of causative genes and pathogenic variants, improved genetic counseling, and the advent of prenatal diagnosis. Translational research has also heralded early phase clinical trials of gene, cell, and protein therapies, as well as enhanced disease models, mechanism-based therapies, and impactful clinical progress.

A clinician's guide to omics resources in dermatology.

With recent advances in high-throughput technologies, we are now in an era where the use of large-scale datasets of biological samples and individual diseases can be analysed using omics methodologies. These include genomics, transcriptomics, proteomics, metabolomics, lipidomics and epigenomics. Omics approaches have been developed to deliver a holistic understanding of systems biology, to identify key biomarkers, and to aid in the interpretation of molecular, biochemical and environmental interactions. Navigating through the plethora of online datasets to find useful and concise information for comparison of data can be complex and overwhelming. The purpose of this article is to review the current repositories and databases, and to evaluate their application in dermatological research and their relevance to clinical practice. For this study, an extensive review of online platforms used in dermatology research was undertaken. Online resources for genetic disease information, genetic disease connection platforms for patients and researchers, clinical interpretation of variants, genome and DNA databases, and omics data repositories and resources were collected. This study provides a comprehensive overview of relevant databases that will aid clinicians and scientists using omics data in dermatology.

Single-cell transcriptomics in human skin research: available technologies, technical considerations and disease applications.

Single-cell technologies have revolutionized research in the last decade, including for skin biology. Single-cell RNA sequencing has emerged as a powerful tool allowing the dissection of human disease pathophysiology at unprecedented resolution by assessing cell-to-cell variation, facilitating identification of rare cell populations and elucidating cellular heterogeneity. In dermatology, this technology has been widely applied to inflammatory skin disorders, fibrotic skin diseases, wound healing complications and cutaneous neoplasms. Here, we discuss the available technologies and technical considerations of single-cell RNA sequencing and describe its applications to a broad spectrum of dermatological diseases.

The TKFC Ala185Thr variant, reported as 'null' for fructose metabolism, is fully active as triokinase.

TKFC-encoded triokinase catalyses glyceraldehyde phosphorylation in fructose metabolism and favours lipogenesis in mice. In Tkfc knockouts or knockdowns, fructose oxidation predominates over lipogenesis. The highly prevalent human variant Ala185Thr-Triokinase/FMN cyclase (TKFC) has been reported to be 'null' for fructose metabolism, since Ala185-TKFC rescues the mouse TKFC-deficient phenotype, whereas Ala185Thr-TKFC does not. Such report implies that most humans would display a noncanonical fructose metabolism, but it ignores the well-characterized triokinase activity of Ala185Thr-TKFC. Here, earlier evidence is summarized, along with new evidence that both human variants are equally active in yeast. Therefore, future research on triokinase in the context of human fructose metabolism should consider that Ala185Thr-TKFC is not biochemically 'null'.

Transcriptomic profiling of recessive dystrophic epidermolysis bullosa wounded skin highlights drug repurposing opportunities to improve wound healing.

Chronic wounds present a major disease burden in people with recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering skin disorder caused by mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils at the dermal-epidermal junction. Treatment of RDEB wounds is mostly symptomatic, and there is considerable unmet need in trying to improve and accelerate wound healing. In this study, we defined transcriptomic profiles and gene pathways in RDEB wounds and compared these to intact skin in RDEB and healthy control subjects. We then used a reverse transcriptomics approach to discover drugs or compounds, which might restore RDEB wound profiles towards intact skin. Differential expression analysis identified >2000 differences between RDEB wounds and intact skin, with RDEB wounds displaying aberrant cytokine-cytokine interactions, Toll-like receptor signalling, and JAK-STAT signalling pathways. In-silico prediction for compounds that reverse gene expression signatures highlighted methotrexate as a leading candidate. Overall, this study provides insight into the molecular profiles of RDEB wounds and underscores the possible clinical value of reverse transcriptomics data analysis in RDEB, and the potential of this approach in discovering or repurposing drugs for other diseases.