Mutations In Keratin Research Articles

Molecular Modeling of Pathogenic Mutations in the Keratin 1B Domain.

Keratin intermediate filaments constitute the primary cytoskeletal component of epithelial cells. Numerous human disease phenotypes related to keratin mutation remain mechanistically elusive. Our recent crystal structures of the helix 1B heterotetramer from keratin 1/10 enabled further investigation of the effect of pathologic 1B domain mutations on keratin structure. We used our highest resolution keratin 1B structure as a template for homology-modeling the 1B heterotetramers of keratin 5/14 (associated with blistering skin disorders), keratin 8/18 (associated with liver disease), and keratin 74/28 (associated with hair disorder). Each structure was examined for the molecular alterations caused by incorporating pathogenic 1B keratin mutations. Structural modeling indicated keratin 1B mutations can harm the heterodimer interface (R265PK5, L311RK5, R211PK14, I150VK18), the tetramer interface (F231LK1, F274SK74), or higher-order interactions needed for mature filament formation (S233LK1, L311RK5, Q169EK8, H128LK18). The biochemical changes included altered hydrophobic and electrostatic interactions, and altered surface charge, hydrophobicity or contour. Together, these findings advance the genotype-structurotype-phenotype correlation for keratin-based human diseases.

Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migration.

Keratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell-extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell-ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.

First person – Sachiko Fujiwara

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Sachiko Fujiwara is first author on ‘Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migration’, published in JCS. Sachiko conducted the research described in this article while a Postdoctoral fellow in Thomas M. Magin's lab at Institute of Biology, Division of Cell & Developmental Biology, Leipzig University, Germany. She is now an assistant professor in the lab of Kazunori Imaizumi at the Graduate School of Biomedical & Health Sciences, Hiroshima University, Japan, investigating the physiological roles of cytoskeletons.

The curly coat phenotype of the Ural Rex feline breed is associated with a mutation in the lipase H gene.

Mutations in lipase H (LIPH) and lysophosphatidic acid receptor 6 (LPAR6), which are essential for the lysophosphatidic acid (LPA) signalling pathway, are associated with hypotrichosis and wooly hair in humans. Mutations in LPAR6 and keratin 71 (KRT71), result in unusual fur growth and hair structure in several cat breeds (Cornish Rex, Devon Rex and Selkirk Rex). Here, we performed target sequencing of the LIPH, LPAR6 and KRT71 genes in six cat breeds with specific hair-growth phenotypes. A LIPH genetic variant (LIPH:c.478_483del; LIPH:p.Ser160_Gly161del) was found in Ural Rex cats with curly coats from Russia, but was absent in all other cat breeds tested. In silico three-dimensional analysis of the LIPH mutant protein revealed a contraction of the α3-helix structure in the enzyme phospholipid binding site that may affect its activity.

Trp53 Mutation in Keratin 5 (Krt5)-Expressing Basal Cells Facilitates the Development of Basal Squamous-Like Invasive Bladder Cancer in the Chemical Carcinogenesis of Mouse Bladder

The biological processes of urothelial carcinogenesis are not fully understood, particularly regarding the relationship between specific genetic events, cell of origin, and molecular subtypes of subsequent tumors. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced mouse bladder cancer is widely accepted as a useful model that recapitulates the pathway of human bladder tumorigenesis from dysplasia to invasive cancer via carcinoma in situ. However, the long and variable time of tumorigenesis often hinders efficient preclinical or translational research. We hypothesized that Trp53 mutation in specific types of urothelial cells facilitates efficient development of clinically relevant bladder cancer. Using lineage tracing, we showed that Trp53 mutation in Krt5-expressing cells resulted in more efficient tumorigenesis of mouse muscle-invasive bladder cancer (MIBC) with squamous differentiation compared with Trp53 mutation in Upk2-expressing cells, or wild-type or hemizygous Trp53 in the entire urothelium. Mouse MIBC that developed at 24 weeks of BBN treatment showed morphologic and genetic similarities to the basal squamous subtypes of human MIBC, irrespective of pre-induction of Trp53 mutation or whether the cell of origin was Krt5- or Upk2-expressing cells. Our findings suggest that intermediate cells as well as basal cells also can give rise to basal-like MIBC, with pre-induction of Trp53 mutation accelerating MIBC. Thus, in BBN chemical carcinogenesis, pre-induction of Trp53 mutation in basal cells facilitates efficient modeling of the basal squamous subtype of human MIBC.

Keratin Dynamics and Spatial Distribution in Wild-Type and K14 R125P Mutant Cells-A Computational Model.

Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell’s interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape.

Mutations in KRT10 in epidermolytic acanthoma.

Epidermolytic acanthoma (EA) is a rare acquired lesion demonstrating a characteristic histopathological pattern of epidermal degeneration referred to as epidermolytic hyperkeratosis (EHK). On histopathological analysis, EA appears nearly identical to inherited EHK-associated dermatoses such as epidermolytic ichthyosis and ichthyosis bullosa of Siemens. While it has been speculated that EA is caused by mutations in KRT10, KRT1, or KRT2 found in these inherited dermatoses, none have yet been identified. Herein, we aim to identify the contributions of keratin mutations to EA. Using genomic DNA extracted from paraffin-embedded samples from departmental archives, we evaluated a discovery cohort using whole-exome sequencing (WES) and assessed remaining samples using Sanger sequencing screening and restriction fragment length polymorphism (RFLP) analysis. DNA from 16/20 cases in our sample was of sufficient quality for polymerase chain reaction amplification. WES of genomic DNA from lesional tissue revealed KRT10 c.466C > T, p.Arg156Cys mutations in 2/3 samples submitted for examination. RFLP analysis of these samples as well as eight additional samples confirmed the mutations identified via WES and identified four additional cases with Arg156 mutations. In sum, 6/11 screened cases showed hotspot mutation in KRT10. Hotspot mutations in the Arg156 position of KRT10, known to cause epidermolytic ichthyosis, also underlie EA.

Epidermolysis Bullosa Simplex Keratinocytes Show Disturbed Mitochondrial Positioning and Activity

Keratin cytoskeletal proteins protect the epidermis against various stresses and participate in the regulation of fundamental cellular processes, such as growth, differentiation, and metabolism. The increasing complexity of keratin functions is underscored by consequences resulting from mutations in keratin 5 and keratin 14 (K14), which cause the skin-blistering disease epidermolysis bullosa simplex (EBS) by promoting the collapse of the keratin cytoskeleton into cytoplasmic protein aggregates (Kiritsi and Nyström, 2018).

Revisiting pachyonychia congenita: a case-cohort study of 815 patients.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders caused by mutations in one of five keratin genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17). The establishment of an international registry containing clinical and molecular data led to the development of a disease classification based on the mutant gene and associated features. To harness the same resource to clarify the prevalence of PC-associated clinical features, delineate phenotype-genotype correlations and identify prognostic features for disease severity. In total, 815 individuals with confirmed keratin mutations registered in the International Pachyonychia Congenita Research Registry were surveyed for clinical findings associated with PC. Data were analysed using various statistical methods, including the Student's t-test, χ2 -test and anova tests for differences in means/proportions. Spearman correlation and logistic regression were used for phenotype-genotype correlations. KRT6A mutations were associated with oral leucokeratosis, hoarseness, youngest age or highest number of fingernails/toenails involved, and use of walking aids. KRT17 mutations were most commonly associated with cysts and natal teeth. Using logistic regression, we found that oral leucokeratosis was correlated with earlier toenail involvement, walking aids, nursing difficulties and hoarseness. Cysts were correlated with oral leucokeratosis, natal teeth and ear wax. Natal teeth predicted earlier toenail involvement, walking difficulties and cyst formation. Hoarseness was correlated with an increased number of involved fingernails. Here, we establish phenotype-genotype correlations in the largest cohort of patients with PC described to date and reveal novel and clinically useful predictors of disease course and manifestations. What's already known about this topic? Pachyonychia congenita (PC) is a group of autosomal dominant disorders caused by mutations in one of five keratin genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17). The main clinical features are nail dystrophy, palmoplantar keratoderma, oral leucokeratosis and cysts. The establishment of an international registry containing the clinical and molecular data of patients with PC led to the development of a disease classification based on the mutant gene and associated features. What does this study add? Data were collected via an international registry to clarify the prevalence of PC-associated clinical features, delineate phenotype-genotype correlations and identify prognostic features for disease severity. This is the largest cohort of patients with PC described to date. The earliest clinical manifestations of PC are nail dystrophy and palmoplantar keratoderma. Diagnosis can be suspected and confirmed in preschool years. Painful plantar keratoderma has the most profound and debilitating effect on quality of life and daily function. Linked Editorial: Steele and O'Toole. Br J Dermatol 2020; 182:521-522. Linked Comment: Mordaunt. Br J Dermatol 2020; 182:537.

443 Mechanisms of Keratinocyte-Melanoma Cell Interaction

Malignant melanoma is the most aggressive form of human skin cancer and shows an increasing incidence. The transformation from a normal melanocyte to a melanoma cell is a stepwise process and the complex mechanisms during early stages of tumor development remain incompletely understood. Based on the increased tumor incidence in Epidermolysis bullosa simplex (EBS), we hypothesize that keratinocytes affect early melanoma development through direct interactions or via indirect mechanisms involving cell-cell contacts, cytoskeletal proteins and by secretion of growth factors. The aim of this project is to characterize underlying mechanisms by which mutations in keratinocyte cytoskeletal proteins affect proliferation, cell-cell-contacts, adhesion and migration of melanoma cells. To determine the influence of keratin mutations on the proliferation and migration of melanoma cells, keratinocytes with mutations associated with EBS were co-cultured with melanoma cells. This revealed an increased proliferation of melanoma cells co-cultured with keratinocytes expressing EBS-associated keratin 5- and keratin 14-mutants in comparison to wild type keratinocytes. In this setting, migration of melanoma cells remained unaffected. Epidermal keratinocytes from regions bordering the tumour were collected from human malignant melanoma samples using laser capture microdissection to identify novel keratinocyte mutations, followed by whole-exome sequencing. Preliminary data suggest somatic mutations in genes related to cell adhesion and cell structure such as FAT1/2, FLG2, LAMA3, JUP, ITGAE, MUC16 and FN1 could be associated with melanoma development. Taken together, mutations in keratin and additional genes involved in cell-cell interactions and the cytoskeleton exert an influence on melanoma cell proliferation. In future experiments, we will examine whether mutations originating in keratinocytes act on melanoma cells directly or indirectly.

436 Macrophages promote growth of squamous cancer independent of T cells

We assessed the role of macrophages in squamous cell carcinoma (SCC) behavior. We used mouse SCC cells derived from tumors harboring a Kras12D activation mutation and Smad4 deletion in keratin 15 (K15)-positive stem cells, and a human SCC cell line, FaDu. SCC cells were transplanted into immune-compromised or -competent (syngeneic) recipients. After tumors were established, we profiled tumor infiltrating leukocytes using CyTOF. CD45+ leukocytes comprised 10-25% of the total tumor cells, and tumor-associated macrophages (TAMs) were 5-10% of the CD45+ cells. There were typically 2-3 fold more M2 TAMs than M1 TAMs. We used clodronate liposomes to deplete TAMs. We found that the number of TAMs was not affected by the presence of T cells but differed among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and macrophages in the spleen, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation, but exhibited increased apoptosis and reduced vascular density. FLIP (Fas-associated via death domain-like interleukin-1β converting enzyme inhibitory protein), an apoptosis inhibitor abundantly produced in tumor cells and TAMs, was reduced in tumor cells of clodronate treated mice. Reduced FLIP levels correlated with reductions in phosphorylated nuclear NFkB p65 and NFkB inhibitor attenuated FLIP protein levels in SCC cells. Further, TGFb1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition (EMT) or TGFb-mediated angiogenesis in endothelial cells. Consequently, metastasis was not significantly reduced by macrophage reduction. However, reduced pSmad3 correlated with reduction of its transcriptional target, vascular endothelial growth factor A (VEGFA), in clodronate-treated tumor cells. Taken together, our study revealed that macrophages contribute to SCC expansion through interactions with tumor cells but are dispensable for SCC metastasis. Our study provides novel insights into understanding the contributions and limitations of TAMs in SCC progression.

087 Revisiting pachyonychia congenita: a case cohort study in 815 patients

Pachyonychia congenita (PC) is a group of autosomal dominant disorders caused by mutations in one of 5 keratin genes (KRT6A, KRT6B, KRT6C, KRT16, or KRT17). The establishment of an international registry containing both clinical and molecular data led to the development of a disease classification based on the mutant gene and associated features. Here we harnessed the same resource to clarify the prevalence of PC-associated clinical features, delineate phenotype-genotype correlations and identify prognostic features for disease severity. 815 individuals with confirmed keratin mutations registered in the International Pachyonychia Congenita Research Registry (IPCRR) were surveyed for clinical findings associated with PC. Data were analyzed using Student T-test, Chi-Square, ANOVA tests (for differences in means/proportions), Spearman correlation and logistic regression (for phenotype-genotype correlation). KRT6A mutations were most common and evident in 40% of cases. Plantar keratoderma and toenail dystrophy were the most common clinical findings, with plantar keratoderma most significantly affecting patient’s quality of life. KRT6A mutations were associated with young age of onset, high number of fingernails/toenails involvement, oral leukokeratosis and hoarseness. Cysts and natal teeth were most common among patients carrying KRT17 mutations. Logistic regression models revealed that oral leukokeratosis correlated with earlier toenail involvement, walking aids use, nursing difficulties and hoarseness. Natal teeth and oral leukokeratosis were inversely correlated one relative to the other, while natal teeth and cysts positively correlated one with the other. Hoarseness predicted an increased number of involved fingernails. Our study reveals novel and clinically useful prognostic predictors in PC, and more generally demonstrates the importance of large registry-based careful studies in rare disorders.

Keratin 6a mutations lead to impaired mitochondrial quality control.

Epidermal differentiation is a multilevel process in which keratinocytes need to lose their organelles, including their mitochondria, by autophagy. Disturbed autophagy leads to thickening of the epidermis as seen in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17. To ask if mitophagy, the selective degradation of mitochondria by autophagy, is disturbed in PC and, if so, at which stage. Immortalized keratinocytes derived from patients with PC were used in fluorescence-based and biochemical assays to dissect the different steps of mitophagy. PC keratinocytes accumulated old mitochondria and displayed disturbed clearance of mitochondria after mitochondrial uncoupling. However, early mitophagy steps and autophagosome formation were not affected. We observed that autolysosomes accumulate in PC and are not sufficiently recycled. We propose an influence of keratins on autolysosomal degradation and recycling. What's already known about this topic? Terminal epidermal differentiation is a multistep process that includes the elimination of cellular components by autophagy. Autophagy-impaired keratinocytes have been shown to result in thickening of epidermal layers. Hyperkeratosis also occurs in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17. What does this study add? Keratins contribute to mitochondrial quality control as well as maintenance of mitochondria-endoplasmic reticulum contact sites. Keratins influence autolysosomal maturation or reformation. What is the translational message? Overaged mitochondria and autolysosomes accumulate in PC. Mutations in keratin 6a lead to severely impaired mitophagy, which might contribute to PC pathogenesis.

Liver disease–associated keratin 8 and 18 mutations modulate keratin acetylation and methylation

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

Macrophages Promote Growth of Squamous Cancer Independent of T cells

Oral cancers, primarily squamous cell carcinomas (SCCs), progress either slowly or aggressively. Here we assessed the role of macrophages in SCC behavior. We used mouse SCC cells derived from tumors harboring a KrasG12D activation mutation and Smad4 deletion in keratin 15–positive stem cells and a human oral SCC cell line, FaDu, which has NRAS amplification and SMAD4 deletion. SCC cells were transplanted into immune-compromised or immune-competent (syngeneic) recipients. After tumors were established, we used clodronate liposomes to ablate macrophages. We found that the number of tumor-associated macrophages (TAMs) was not affected by the presence of T cells but differed considerably among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and splenic macrophages, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation but did exhibit increased apoptosis and reduced vascular density. FLIP (Fas-associated via death domain-like interleukin 1β–converting enzyme inhibitory protein), an apoptosis inhibitor abundantly produced in tumor cells and TAMs, was reduced in tumor cells of clodronate-treated mice. Reduced FLIP levels correlated with reductions in phosphorylated nuclear NFκB p65 and NFκB inhibitor attenuated FLIP protein levels in SCC cells. Furthermore, TGFβ1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition or TGFβ-mediated angiogenesis in endothelial cells. Consequently, metastasis was not significantly reduced by macrophage reduction. However, reduced pSmad3 correlated with reduction of its transcriptional target, vascular endothelial growth factor A, in clodronate-treated tumor cells, which correlated with reduced vascular density in clodronate-treated tumors. Taken together, our study revealed that macrophages contribute to SCC expansion through interactions with tumor cells but are dispensable for SCC metastasis. Our study provides novel insights into understanding the contributions and limitations of TAMs in SCC progression.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BackgroundMissense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch.MethodsWe extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA).Findings4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1β expression- but also showed activation of Wnt/β-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner.InterpretationTogether, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

The role of keratins in the digestive system: lessons from transgenic mouse models.

Keratins are the largest subfamily of intermediate filament proteins. They are either type I acidic or type II basic keratins. Keratins form obligate heteropolymer in epithelial cells and their expression patterns are tissue-specific. Studies have shown that keratin mutations are the cause of many diseases in humans or predispose humans to acquiring them. Using mouse models to study keratin-associated human diseases is critical, because they allow researchers to get a better understanding of these diseases and their progressions, and so many such studies have been conducted. Acknowledging the importance, researches with genetically modified mice expressing human disease-associated keratin mutants have been widely done. Numerous studies using keratin knockout mice, keratin-overexpressed mice, or transgenic mice expressing keratin mutants have been conducted. This review summarizes the mouse models that have been used to study type I and type II keratin expression in the digestive organs, namely, the liver, pancreas, and colon.

Decreased levels of keratin 8 sensitize mice to streptozotocin-induced diabetes.

AimDiabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β‐cell keratins, and knockout of K8 (K8−/−) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β‐cell stress and that decreased K8 levels contribute to diabetes susceptibility.MethodsK8‐heterozygous knockout (K8+/−) and wild‐type (K8+/+) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)‐induced β‐cell stress and T2D high‐fat diet (HFD).ResultsMurine K8+/− endocrine islets express ~50% less K8/K18 compared with K8+/+. The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, β‐cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8+/− mice after low‐dose/chronic STZ treatment, and STZ causes more β‐cell damage and polyuria in K8+/− compared with K8+/+. K8 appears upregulated 5 weeks after STZ treatment in K8+/+ islets but not in K8+/−. K8+/− mice showed no major susceptibility risk to HFD compared to K8+/+.ConclusionPartial K8 deficiency reduces β‐cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD.

Mutational analysis of epidermal and hyperproliferative type I keratins in mild and moderate psoriasis vulgaris patients: a possible role in the pathogenesis of psoriasis along with disease severity

BackgroundMutations in keratin proteins have been vastly associated with a wide array of genodermatoses; however, mutations of keratins in psoriasis have not been fully investigated. The main aim of the current research was to identify the mutation in K14, K10, K16, and K17 genes in two stages of psoriasis patients.MethodsNinety-six psoriatic skin biopsies were collected. mRNA transcript of K14, K10, K16, and K17 was prepared, amplified, and sequenced. Sanger sequences of all keratins were further validated for mutational analysis using Mutation Surveyor and Alamut Visual. Then, in silico analysis of protein stability and protein and gene expression of all keratins was performed and validated.ResultsOut of 44 mutations, about 75% of keratins are highly pathogenic and deleterious. Remaining 25% mutations are less pathogenic and tolerated in nature. In these 33 deleterious mutations were immensely found to decrease keratin protein stability. We also found a correlation between keratin and Psoriasis Area and Severity Index score which added that alteration in keratin gene in skin causes severity of psoriasis.ConclusionsWe strongly concluded that acanthosis and abnormal terminal differentiation was mainly due to the mutation in epidermal keratins. In turn, disease severity and relapsing of psoriasis are mainly due to the mutation of hyperproliferative keratins. These novel keratin mutations in psoriatic epidermis might be one of the causative factors for psoriasis.

752 Simultaneous gene-editing and reprogramming of epidermolysis bullosa simplex fibroblasts

Epidermolysis bullosa simplex (EBS) is an autosomal dominant skin fragility disorder caused by mutations in either Keratin (K) 5 or K14. Although current therapy for EBS is limited to wound care, advances in reprogramming somatic cells into induced pluripotent stem cells (iPSC) offer the possibility of developing new approaches for EBS treatment. The iPSC-based therapeutic approach for EBS relies on the generation of patient-specific iPSCs, which then undergo genetic editing and differentiation into skin cells suitable for transplantation. One of the main hurdles in advancing the iPSC-based therapy into the clinic is the complexity of a multi-step manufacturing process to produce genetically corrected iPSC-derived epidermal sheets. To reduce the manufacturing complexity of an iPSC-based therapy for EBS, we have combined our previously presented high-efficiency RNA-based reprogramming approach with a specific CRISPR/Cas9-mediated knock-out of the mutant allele of the K14 gene into a one-step procedure. We established a fibroblast line from an EBS patient carrying a hot-spot heterozygous C373T mutation at codon 125 in K14 and successfully generated multiple isogenic EBS iPSCs lines with a specific knock out of the mutant K14 allele (KO EBS iPSC) within 4 weeks of initiating simultaneous gene editing and reprogramming. Simultaneous reprogramming and gene correction not only reduces the complexity of the iPSC-based therapy but also improves safety of the approach by avoiding lengthy cell culture periods, drug selection, and multiple sub-cloning steps. We are currently differentiating these KO EBS iPSCs into keratinocytes to determine if these gene edited iPSC-derived keratinocytes can be used for EBS therapy by employing a mouse xenograft model.