Filaggrin Mutation Status and Prevention of Atopic Dermatitis with Maternal Probiotic Supplementation.

The World Allergy Organization recommends probiotics in the prevention of atopic dermatitis in high-risk populations. Mutations in the filaggrin gene (FLG) result in an increased risk of atopic dermatitis through disruption of the skin keratin layer. This exploratory study investigated whether the preventive effect of maternal probiotics was evident in children with and without FLG mutations. DNA was collected from children (n = 228) from the Probiotic in the Prevention of Allergy among Children in Trondheim (ProPACT) study. Samples were analysed for 3 common FLG mutations (R501X, R2447X, and 2282del4). Overall, 7% of children had heterozygous FLG mutations; each child had only one of the 3 mutations. Mutation status had no association with atopic dermatitis (RR = 1.1; 95% CI 0.5 to 2.3). The risk ratio (RR) for having atopic dermatitis following maternal probiotics was 0.6 (95% CI 0.4 to 0.9) and RR was similar if the child expressed an FLG mutation (RR = 0.6; 95% CI 0.1 to 4.1) or wildtype FLG (RR = 0.6; 95% CI 0.4 to 0.9). The preventive  effect of probiotics for atopic dermatitis was also evident in children without FLG mutation. Larger confirmatory studies are needed.

Keratinocyte-derived small extracellular vesicles supply antigens for CD1a-resticted T cells and promote their type 2 bias in the context of filaggrin insufficiency.

Introduction: Exosome-enriched small extracellular vesicles (sEVs) are nanosized organelles known to participate in long distance communication between cells, including in the skin. Atopic dermatitis (AD) is a chronic inflammatory skin disease for which filaggrin (FLG) gene mutations are the strongest genetic risk factor. Filaggrin insufficiency affects multiple cellular function, but it is unclear if sEV-mediated cellular communication originating from the affected keratinocytes is also altered, and if this influences peptide and lipid antigen presentation to T cells in the skin. Methods: Available mRNA and protein expression datasets from filaggrin-insufficient keratinocytes (shFLG), organotypic models and AD skin were used for gene ontology analysis with FunRich tool. sEVs secreted by shFLG and control shC cells were isolated from conditioned media by differential centrifugation. Mass spectrometry was carried out for lipidomic and proteomic profiling of the cells and sEVs. T cell responses to protein, peptide, CD1a lipid antigens, as well as phospholipase A2-digested or intact sEVs were measured by ELISpot and ELISA. Results: Data analysis revealed extensive remodeling of the sEV compartment in filaggrin insufficient keratinocytes, 3D models and the AD skin. Lipidomic profiles of shFLGsEV showed a reduction in the long chain (LCFAs) and polyunsaturated fatty acids (PUFAs; permissive CD1a ligands) and increased content of the bulky headgroup sphingolipids (non-permissive ligands). This resulted in a reduction of CD1a-mediated interferon-γ T cell responses to the lipids liberated from shFLG-generated sEVs in comparison to those induced by sEVs from control cells, and an increase in interleukin 13 secretion. The altered sEV lipidome reflected a generalized alteration in the cellular lipidome in filaggrin-insufficient cells and the skin of AD patients, resulting from a downregulation of key enzymes implicated in fatty acid elongation and desaturation, i.e., enzymes of the ACSL, ELOVL and FADS family. Discussion: We determined that sEVs constitute a source of antigens suitable for CD1a-mediated presentation to T cells. Lipids enclosed within the sEVs secreted on the background of filaggrin insufficiency contribute to allergic inflammation by reducing type 1 responses and inducing a type 2 bias from CD1a-restricted T cells, thus likely perpetuating allergic inflammation in the skin.

Comparative genomics of sirenians reveals evolution of filaggrin and caspase-14 upon adaptation of the epidermis to aquatic life.

The mammalian epidermis has evolved to protect the body in a dry environment. Genes of the epidermal differentiation complex (EDC), such as FLG (filaggrin), are implicated in the barrier function of the epidermis. Here, we investigated the molecular evolution of the EDC in sirenians (manatees and dugong), which have adapted to fully aquatic life, in comparison to the EDC of terrestrial mammals and aquatic mammals of the clade Cetacea (whales and dolphins). We show that the main subtypes of EDC genes are conserved or even duplicated, like late cornified envelope (LCE) genes of the dugong, whereas specific EDC genes have undergone inactivating mutations in sirenians. FLG contains premature stop codons in the dugong, and the ortholog of human CASP14 (caspase-14), which proteolytically processes filaggrin, is pseudogenized in the same species. As FLG and CASP14 have also been lost in whales, these mutations represent convergent evolution of skin barrier genes in different lineages of aquatic mammals. In contrast to the dugong, the manatee has retained functional FLG and CASP14 genes. FLG2 (filaggrin 2) is truncated in both species of sirenians investigated. We conclude that the land-to-water transition of sirenians was associated with modifications of the epidermal barrier at the molecular level.

Association between polymorphisms and atopic dermatitis susceptibility: A systematic review and meta-analysis.

AIM: Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease that is closely linked to genetic factors. Previous studies have revealed numerous single nucleotide polymorphisms (SNPs) that been related to susceptibility to AD; however, the results are conflicting. Therefore, a meta-analysis was conducted to assess the associations of these polymorphisms and AD risk. MATERIAL AND METHODS: PubMed, Web of Science, Embase, Cochrane Library, and China National Knowledge Infrastructure databases were retrieved to identify eligible studies, with selected polymorphisms being reported in a minimum of three separate studies. The Newcastle-Ottawa Scale (NOS) was used to evaluate study quality. Review Manager 5.3 and STATA 14.0 were used to perform the meta-analysis. RESULTS: After screening, 64 studies involving 13 genes (24 SNPs) were selected for inclusion in the meta-analysis. Nine SNPs were positively correlated with AD susceptibility [filaggrin (FLG) R501X, FLG 2282del4, chromosome 11q13.5 rs7927894, interleukin (IL)-17A rs2275913, IL-18 -137 G/C, Toll-like receptor 2 (TLR2) rs5743708, TLR2 A-16934 T, serine protease inhibitor Kazal type-5 (SPINK5) Asn368Ser, interferon-γ (IFN-γ) T874A] and one was negatively associated with AD susceptibility (IL-4 -1098 T/G). The 14 remaining SNPs were not significantly associated with AD susceptibility. CONCLUSIONS: Nine SNPs that may be risk factors and one SNP that may be a protective factor for AD were identified, providing a reference for AD prediction, prevention, and therapy.

Decreased TET2/5-hmC reduces the integrity of the epidermal barrier via epigenetic dysregulation of filaggrin in psoriatic lesions.

BACKGROUND: TET2 participates in tumor progression and intrinsic immune homeostasis via epigenetic regulation. TET2 has been reported to be involved in maintaining epithelial barrier homeostasis and inflammation. Abnormal epidermal barrier function and TET2 expression have been detected in psoriatic lesions. However, the mechanisms underlying the role of TET2 in psoriasis have not yet been elucidated. OBJECTIVE: To define the role of TET2 in maintaining epithelial barrier homeostasis and the exact epigenetic mechanism in the dysfunction of the epidermal barrier in psoriasis. METHODS: We analyzed human psoriatic skin lesions and datasets from the GEO database, and detected the expression of TET2/5-hmC together with barrier molecules by immunohistochemistry. We constructed epidermal-specific TET2 knockout mice to observe the effect of TET2 deficiency on epidermal barrier function via toluidine blue penetration assay. Further, we analyzed changes in the expression of epidermal barrier molecules by immunofluorescence in TET2-specific knockout mice and psoriatic model mice. RESULTS: We found that decreased expression of TET2/5-hmC correlated with dysregulated barrier molecules in human psoriatic lesions. Epidermal-specific TET2 knockout mice showed elevated transdermal water loss associated with abnormal epidermal barrier molecules. Furthermore, we observed that TET2 knockdown in keratinocytes reduced filaggrin expression via filaggrin promoter methylation. CONCLUSION: Aberrant epidermal TET2 affects the integrity of the epidermal barrier through the epigenetic dysregulation of epidermal barrier molecules, particularly filaggrin. Reduced TET2 expression is a critical factor contributing to an abnormal epidermal barrier in psoriasis.

Infant lung function and early skin barrier impairment in the development of asthma at age 3 years.

BACKGROUND: Largely unexplored, we investigated if lower lung function, impaired skin barrier function by transepidermal water loss (TEWL), eczema, and filaggrin (FLG) mutations in infancy were associated with asthma in early childhood. METHODS: From the factorially designed randomized controlled intervention study PreventADALL, we evaluated 1337/2394 children from all randomization groups with information on asthma at age 3 years, and at age 3 months either lung function, TEWL, eczema, and/or FLG mutations. Lower lung function was defined as the time to peak tidal expiratory flow to expiratory time (tPTEF /tE ) <0.25, and skin barrier impairment as a high TEWL >9.50 g/m2 /h. Eczema was clinically observed, and DNA genotyped for FLG mutations. Asthma was defined as asthma-like symptoms (≥3 episodes of bronchial obstruction) between age 2-3 years as well as a history of doctor-diagnosed asthma and/or asthma medication use. Associations were analyzed in logistic regression models, presented with adjusted ORs (aOR) and 95% confidence intervals (CI). RESULTS: Lower lung function and skin barrier impairment were associated with asthma in general; aOR (95% CI) 5.4 (2.1, 13.7) and 1.6 (1.1, 2.5), while eczema and FLG mutations were associated with asthma in children with atopic dermatitis or allergic sensitization only. Stratifying for sex, the risk of asthma was only increased in boys with lower lung function; aOR (95% CI) 7.7 (2.5, 23.6), and in girls with FLG mutations; aOR (95% CI) 3.5 (1.5, 8.2). CONCLUSION: Lower lung function and impaired skin barrier function in infancy may increase the risk of asthma at age 3 years.

Filaggrin and beyond: New insights into the skin barrier in atopic dermatitis and allergic diseases, from genetics to therapeutic perspectives.

Atopic dermatitis (AD) is the most common inflammatory skin disease worldwide, affecting 20% of children and 5% of adults. One critical component in the pathophysiology of AD is the epidermal skin barrier, with its outermost layer, the stratum corneum (SC), conferring biochemical properties that enable resilience against environmental threats and maintain homeostasis. The skin barrier may be conceptualized as a key facilitator of complex interactions between genetics, host immunity, the cutaneous microbiome, and environmental exposures. The key genetic risk factor for AD development and persistence is a loss-of-function mutation in FLG, with recent advances in genomics focusing on rare variant discovery, establishment of pathogenic mechanisms, and exploration of the role of other epidermal differentiation complex gene variants in AD. Aberrant type 2 inflammatory responses down-regulate the transcription of key epidermal barrier genes, alter the composition of SC lipids, and induce further injury through a neurocutaneous feedback loop and the itch-scratch cycle. The dysbiotic epidermis exhibits reduced bacterial diversity and enhanced colonization with Staphylococcus and Malassezia species, which contribute to both direct barrier injury through the action of bacterial toxins and perpetuation of the inflammatory cascades. Enhanced understanding of each of the pathogenic mechanisms underpinning barrier disruption has led to the development of novel topical and systemic molecules, including interleukin (IL)-4Ra, IL-13, PDE4, and Janus-associated kinase inhibitors, whose clinical effectiveness exceeds conventional treatment modalities. In this narrative review, we aim to summarize the current understanding of the above-mentioned pathophysiological and therapeutic mechanisms, with a focus on the genetic, cellular, and molecular mechanisms underpinning AD development.

Inflammation-related proteins in blood after dermal exposure to some common chemicals depend on the skin barrier gene filaggrin - a human experimental study.

Filaggrin (FLG), a skin barrier protein, is associated with higher dermal uptake of some chemicals in carriers of loss-of-function (null) mutations. This study investigates FLG mutations and systemic effects following dermal exposure to chemicals. Individuals (n = 23 FLG null, n = 31 FLG wt) were simultaneously exposed to pyrimethanil, pyrene, oxybenzone, and nickel ions for 4 h. Pre- and post-exposure, 25-hydroxyvitamin D3 (25(OH)D3, LC-MS/MS) and 92 inflammation-related proteins (proximity-extension assay) were measured. FLG null carriers exhibited significantly higher 25(OH)D3 concentrations than wt carriers, both pre- and post-exposure. Eleven proteins differed in abundance post- vs pre-exposure among FLG null carriers, and 22 proteins among wt carriers (three proteins overlapped). Twelve proteins showed median differences (post- vs pre-exposure) between FLG null and wt carriers. Overall, FLG null carriers showed an increase, while FLG wt carriers showed a decrease in inflammation-related proteins. These findings suggest FLG-dependent differences in susceptibility to systemic effects following simultaneous dermal chemical exposure.

Identification of eczema clusters and their association with filaggrin and atopic comorbidities: analysis of five birth cohorts.

BACKGROUND: Longitudinal modelling of the presence/absence of current eczema through childhood has identified similar phenotypes, but their characteristics often differ between studies. OBJECTIVES: To demonstrate that a more comprehensive description of longitudinal pattern of symptoms may better describe trajectories than binary information on eczema presence. METHODS: We derived six multidimensional variables of eczema spells from birth to 18 years of age (including duration, temporal sequencing and the extent of persistence/recurrence). Spells were defined as consecutive observations of eczema separated by no eczema across 5 epochs in five birth cohorts: infancy (first year); early childhood (age 2-3 years); preschool/early school age (4-5 years); middle childhood (8-10 years); adolescence (14-18 years). We applied Partitioning Around Medoids clustering on these variables to derive clusters of the temporal patterns of eczema. We then investigated the stability of the clusters, within-cluster homogeneity and associated risk factors, including FLG mutations. RESULTS: Analysis of 7464 participants with complete data identified five clusters: (i) no eczema (51.0%); (ii) early transient eczema (21.6%); (iii) late-onset eczema (LOE; 8.1%); (iv) intermittent eczema (INT; 7.5%); and (v) persistent eczema (PE; 11.8%). There was very-high agreement between the assignment of individual children into clusters when using complete or imputed (n = 15 848) data (adjusted Rand index = 0.99; i.e. the clusters were very stable). Within-individual symptom patterns across clusters confirmed within-cluster homogeneity, with consistent patterns of symptoms among participants within each cluster and no overlap between the clusters. Clusters were characterized by differences in associations with risk factors (e.g. parental eczema was associated with all clusters apart from LOE; sensitization to inhalant allergens was associated with all clusters, with the highest risk in the PE cluster). All clusters apart from LOE were associated with FLG mutations. Of note, the strongest association was for PE [relative risk ratio (RRR) 2.70, 95% confidence interval (CI) 2.24-3.26; P < 0.001] followed by INT (RRR 2.29, 95% CI 1.82-2.88; P < 0.001). CONCLUSIONS: Clustering of multidimensional variables identified stable clusters with different genetic architectures. Using multidimensional variables may capture eczema development and derive stable and internally homogeneous clusters. However, deriving homogeneous symptom clusters does not necessarily mean that these are underpinned by completely unique mechanisms.

Filaggrin gene variants among Saudi patients with ichthyosis vulgaris.

Ichthyoses are a heterogeneous group of cornification disorders. The most common form of ichthyoses is ichthyosis vulgaris (IV) ([OMIM] #146,700), which can be inherited as autosomal semi-dominant mutation in the filaggrin gene (FLG). We present the findings of a study involving 35 Saudi patients with a clinical diagnosis of ichthyosis vulgaris. For identifying the pathogenic mutation of their disease, we used Sanger sequencing analysis of the extracted DNA samples. We also identified the underlying 22 FLG variants, which have been seen before. However, the detected mutations do not involve the common p.R501* c. 2282del4 mutations reported in European populations. Indeed, we did not identify any statistical influence of the homozygous or heterozygous genotypes on the phenotype severity of the disease.

A New Mouse Model of Giant Axonal Neuropathy with Overt Phenotypes and Neurodegeneration Driven by Neurofilament Disorganization.

Research on pathogenic mechanisms underlying giant axonal neuropathy (GAN), a disease caused by a deficiency of gigaxonin, has been hindered by the lack of appropriate animal models exhibiting substantial symptoms and large neurofilament (NF) swellings, a hallmark of the human disease. It is well established that intermediate filament (IF) proteins are substrates for gigaxonin-mediated degradation. However, it has remained unknown to what extent NF accumulations contribute to GAN pathogenesis. Here, we report the generation of a new mouse model of GAN that is based on crossing transgenic mice overexpressing peripherin (Prph) with mice knockout for Gan The Gan-/-;TgPer mice developed early onset sensory-motor deficits along with IF accumulations made up of NF proteins and of Prph, causing swelling of spinal neurons at a young age. Abundant inclusion bodies composed of disorganized IFs were also detected in the brain of Gan-/-;TgPer mice. At 12 months of age, the Gan-/-;TgPer mice exhibited cognitive deficits as well as severe sensory and motor defects. The disease was associated with neuroinflammation and substantial loss of cortical neurons and spinal neurons. Giant axons (≥160 µm2) enlarged by disorganized IFs, a hallmark of GAN disease, were also detected in dorsal and ventral roots of the Gan-/-;TgPer mice. These results, obtained with both sexes, support the view that the disorganization of IFs can drive some neurodegenerative changes caused by gigaxonin deficiency. This new mouse model should be useful to investigate the pathogenic changes associated with GAN disease and for drug testing.SIGNIFICANCE STATEMENT Research on pathogenic mechanism and treatment of GAN has been hampered by the lack of animal models exhibiting overt phenotypes and substantial neurofilament disorganization, a hallmark of the disease. Moreover, it remains unknown whether neurologic defects associated with gigaxonin deficiency in GAN are because of neurofilament disorganization as gigaxonin may also act on other protein substrates to mediate their degradation. This study reports the generation of a new mouse model of GAN based on overexpression of Prph in the context of targeted disruption of gigaxonin gene. The results support the view that neurofilament disorganization may contribute to neurodegenerative changes in GAN disease. The Gan-/-;TgPer mice provide a unique animal model of GAN for drug testing.

PDE4 inhibition by difamilast regulates filaggrin and loricrin expression via keratinocyte proline-rich protein in human keratinocytes.

BACKGROUND: Difamilast, a topical phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective for treating atopic dermatitis (AD), but the molecular mechanism involved is unclear. Since skin barrier dysfunction including reduced expression of filaggrin (FLG) and loricrin (LOR) contributes to AD development, difamilast treatment may be able to improve this dysfunction. PDE4 inhibition increases transcriptional activity of cAMP-responsive element binding protein (CREB). Therefore, we hypothesized that difamilast may affect FLG and LOR expression via CREB in human keratinocytes. OBJECTIVE: To elucidate the mechanism by which difamilast regulates FLG and LOR expression via CREB in human keratinocytes. METHODS: We analyzed normal human epidermal keratinocytes (NHEKs) treated with difamilast. RESULTS: We observed increases of intracellular cAMP levels and CREB phosphorylation in difamilast (5 µM)-treated NHEKs. Next, we found that difamilast treatment increased mRNA and protein levels of FLG and LOR in NHEKs. Since reduced expression of keratinocyte proline-rich protein (KPRP) is reported to be involved in skin barrier dysfunction in AD, we examined KPRP expression in difamilast-treated NHEKs. We found that difamilast treatment increased mRNA and protein levels of KPRP in NHEKs. Furthermore, KPRP knockdown using siRNA transfection abolished the upregulation of FLG and LOR in difamilast-treated NHEKs. Finally, CREB knockdown canceled the upregulation of FLG, LOR, and KPRP in difamilast-treated NHEKs, indicating that PDE4 inhibition by difamilast treatment positively regulates FLG and LOR expression via the CREB-KPRP axis in NHEKs. CONCLUSION: These findings may provide further guidance for therapeutic strategies in the treatment of AD using difamilast.

Barrier function and ultrastructure characteristics of epidermis in patients with primary cutaneous amyloidosis.

Previous studies on primary cutaneous amyloidosis (PCA) have mainly focused on exploring genetic mutation and components of amyloid in patients with PCA. However, studies on skin barrier function in PCA patients are scarce. Here, we detected the skin barrier function in PCA patients and healthy people by using noninvasive techniques and characterized ultrastructural features of PCA lesions compared with healthy people using transmission electron microscopy (TEM). The expression of proteins related to skin barrier function was examined by immunohistochemistry staining. A total of 191 patients with clinically diagnosed PCA and 168 healthy individuals were enrolled in the study. Our analysis revealed that all investigated lesion areas displayed higher transepidermal water loss and pH values, and lower Sebum levels and stratum corneum hydration levels in PCA patients compared with the same site area in healthy individuals. The TEM results showed that the intercellular spaces between the basal cells were enlarged and the number of hemidesmosomes decreased in PCA lesions. Immunohistochemical staining showed that the expression of integrin α6 and E-cadherin in PCA patients was less than that in healthy controls, while no differences in the expression of loricrin and filaggrin were observed. Our study revealed that individuals with PCA displayed skin barrier dysfunction, which may be related to alterations in epidermal ultrastructure and a decrease in the skin barrier-related protein E-cadherin. However, the molecular mechanisms underlying skin barrier dysfunction in PCA remain to be elucidated.

Filaggrin gene polymorphisms in Indian children with atopic dermatitis: A cross-sectional multicentre study.

Background Filaggrin (FLG) gene encoding the protein filaggrin plays an important role in barrier function of the skin and its alteration is a predisposing factor for atopic dermatitis. FLG gene variants result in absent or decreased filaggrin protein. Worldwide, the prevalence of FLG variants ranges from 14 to 56%. FLG null variants are distinct in each population. Objectives To study the FLG gene polymorphisms in Indian children and attempt a genotype-phenotype correlation in atopic dermatitis. Methods This was a cross-sectional, multicentre study conducted on 75 Indian children. Demographic details, clinical features and identified FLG null variants were recorded. We performed a whole gene sequencing of the entire FLG coding region using next-generation sequencing technology. Results The prevalence of FLG null variants was 34.7%. A total of 20 different FLG loss of function variants in 26 children were documented. Sixteen (80%) variants were novel and four (20%) were previously reported in Asian and European populations. We found a statistically significant association between FLG variants with early age of onset of atopic dermatitis (P = 0.016) and elevated serum IgE levels (P = 0.051). There was no significant difference between atopic dermatitis phenotypes in children having one variant as compared to children harbouring two or more null variants. Limitation Small sample size. Conclusion Our study reports a unique set of FLG variants different from Asian and European populations, with these variants being significantly associated with an early age of onset of atopic dermatitis and elevated serum IgE levels.

Altered expression of S100 fused-type proteins in an atopic dermatitis skin model.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder with elevated interleukin (IL)-4 and IL-13 signatures and extensive barrier dysfunction, which is correlated with the downregulation of filaggrin (FLG). FLG is a member of the S100 fused-type protein family and this family also includes cornulin (CRNN), filaggrin-2 (FLG2), hornerin (HRNR) repetin (RPTN), trichohyalin (TCHH) and trichohyalin-like 1 (TCHHL1). The present study aimed to examine the effects of IL-4 and IL-13 and the downregulation of FLG on the expression of S100 fused-type proteins using a three-dimensional (3D) AD skin model by immunohistochemical study and quantitative polymerase chain reaction. In the 3D AD skin model, which was generated by a stimulation of recombinant IL-4 and IL-13, the expression of FLG, FLG2, HRNR and TCHH was decreased, while that of RPTN was increased in comparison to the 3D control skin. In the FLG knockdown (KD) 3D skin model, which was generated using FLG siRNA, the expression of HRNR was increased. The expression of the other proteins did not differ to a statistically significant extent. The expression of fused-S100 type protein family members may differ in AD skin. This suggests that these proteins play different roles in the pathogenesis of AD.

"In the light of evolution:" keratins as exceptional tumor biomarkers.

Keratins (KRTs) are the intermediate filament-forming proteins of epithelial cells, classified, according to their physicochemical properties, into "soft" and "hard" keratins. They have a key role in several aspects of cancer pathophysiology, including cancer cell invasion and metastasis, and several members of the KRT family serve as diagnostic or prognostic markers. The human genome contains both, functional KRT genes and non-functional KRT pseudogenes, arranged in two uninterrupted clusters on chromosomes 12 and 17. This characteristic renders KRTs ideal for evolutionary studies. Herein, comprehensive phylogenetic analyses of KRT homologous proteins in the genomes of major taxonomic divisions were performed, so as to fill a gap in knowledge regarding the functional implications of keratins in cancer biology among tumor-bearing species. The differential expression profiles of KRTs in diverse types of cancers were investigated by analyzing high-throughput data, as well. Several KRT genes, including the phylogenetically conserved ones, were found to be deregulated across several cancer types and to participate in a common protein-protein interaction network. This indicates that, at least in cancer-bearing species, these genes might have been under similar evolutionary pressure, perhaps to support the same important function(s). In addition, semantic relations between KRTs and cancer were detected through extensive text mining. Therefore, by applying an integrative in silico pipeline, the evolutionary history of KRTs was reconstructed in the context of cancer, and the potential of using non-mammalian species as model organisms in functional studies on human cancer-associated KRT genes was uncovered.