Acral lamellar ichthyosis with amino acid substitution in the C-terminus of keratin 2.

BACKGROUND: Most cases of hereditary ichthyoses present with generalized scaling and skin dryness. However, in some cases skin involvement is restricted to particular body regions as in acral lamellar ichthyosis. OBJECTIVES: We report on the genetic basis of acral ichthyosis in two families presenting with a similar phenotype. METHODS: Genetic testing was performed by targeted next generation sequencing and whole-exome sequencing. For identity-by-descent analysis, the parents were genotyped and data analysis was performed with the Chromosome Analysis Suite Software. RT-PCR with RNA extracted from skin samples was used to analyse the effect of variants on splicing. RESULTS: Genetic testing identified a few heterozygous variants, but only the variant in KRT2 c.1912 T > C, p.Phe638Leu segregated with the disease and remained the strongest candidate. Pairwise identity-by-descent analysis revealed no indication of family relationship. Phenylalanine 638 is the second last amino acid upstream of the termination codon in the tail of K2, and substitution to leucine is predicted as probably damaging. Assessment of the variant is difficult, in part due to the lack of crystal structures of this region. CONCLUSIONS: Altogether, we show that a type of autosomal dominant acral ichthyosis is most probably caused by an amino acid substitution in the C-terminus of keratin 2.

Deep Phenotyping of Superficial Epidermolytic Ichthyosis due to a Recurrent Mutation in KRT2.

Superficial epidermolytic ichthyosis (SEI) is an autosomal dominant inherited ichthyosis. SEI is caused by mutations in KRT2 and frequently shows erythroderma and widespread blistering at birth. We report the clinical manifestations of two patients from a Japanese family with SEI caused by a hotspot mutation, p.Glu487Lys, in KRT2. In addition, we summarize previous reports on SEI patients with the identical mutation. One of the two patients had disease onset at the age of 7 months. The other patient's age of onset is unknown, but it was in childhood. Neither of the two patients showed erythroderma. To perform deep phenotyping, we studied the age of onset and the frequency of erythroderma in 34 reported SEI cases with the p.Glu487Lys mutation, including the present cases. Among the cases with sufficient clinical information, 44.4% of the cases that were due to p.Glu487Lys in KRT2 occurred at birth. Erythroderma was observed in 11.1% of the cases with p.Glu487Lys in KRT2.

Nonsense mutations in KRT1 caused recessive epidermolytic palmoplantar keratoderma with knuckle pads.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is characterized by diffuse hyperkeratosis affecting palms and soles with suprabasal epidermolysis or vacuolar degeneration histopathologically. The disorder is caused by heterozygous mutations in KRT9 or KRT1. Dominant-negative mutations in KRT1 could also result in epidermolytic ichthyosis with EPPK, a more severe entity affecting the entire body. OBJECTIVE: To investigate the genetic basis and pathogenesis of two unrelated patients with EPPK and knuckle pads, both of whom were born to consanguineous parents of Chinese origin. METHODS: Next-generation sequencing was applied to the two patients using genomic DNA extracted from peripheral blood. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western blot (WB) were employed to evaluate mRNA and protein expression level. Ultrastructural changes of skin lesion were analysed using transmission electron microscopy. RESULTS: Two novel homozygous mutations, c.457C>T (p.Gln153*) and c.33C>G (p.Tyr11*) in KRT1, were identified in patients 1 and 2 respectively. The nonsense mutations were predicted to result in nonsense-mediated mRNA decay and absence of keratin 1, which was confirmed in the skin lesions from patient 1. Upregulated keratin 2 was detected both in the affected and unaffected skin samples from patient 1, while the protein abundance and distribution pattern of keratin 10 remained unchanged. An aberrant and clumped staining pattern of keratin 9 was noted in the palmar skin of patient 1. CONCLUSIONS: Homozygous 'knockout' mutations in KRT1 resulted in EPPK with knuckle pads rather than epidermolytic ichthyosis. We speculated that sparing of non-acral skin might be due to compensatory effect of keratin 2 upregulation by forming heterodimer with keratin 10.

Scabies in a 14-year-old girl with superficial epidermolytic ichthyosis.

A 14-year-old girl who reported generalized scaling and hyperkeratosis since age 1 year presented with severe pruritus of several months' duration. Scabies mites were detected, and molecular genetic analysis subsequently revealed a rare pathogenic variant in the keratin 2 (KRT2) gene, confirming a diagnosis of superficial epidermolytic ichthyosis. Treatment with oral ivermectin led to complete remission of symptoms. Disorders of keratinization can mimic clinical signs of scabies, leading to a delay in diagnosis.

Epidermolytic epidermal nevus caused by a somatic mutation in KRT2.

Superficial epidermolytic ichthyosis (formerly Ichthyosis bullosa of Siemens) is an uncommon condition caused by dominant mutations in KRT2 encoding keratin 2. Epidermolytic epidermal nevus due to somatic mutations in KRT2 is even rarer. Here, we report the third case of KRT2-associated epidermal nevus and review the literature.

First Case of KRT2 Epidermolytic Nevus and Novel Clinical and Genetic Findings in 26 Italian Patients with Keratinopathic Ichthyoses.

Keratinopathic ichthyoses (KI) are a clinically heterogeneous group of keratinization disorders due to mutations in KRT1, KTR10, or KRT2 genes encoding keratins of suprabasal epidermis. Characteristic clinical features include superficial blisters and erosions in infancy and progressive development of hyperkeratosis. Histopathology shows epidermolytic hyperkeratosis. We describe the clinical, histopathological, and molecular findings of a series of 26 Italian patients from 19 unrelated families affected with (i) epidermolytic ichthyosis due to KRT1 or KRT10 mutations (7 and 9 cases, respectively); (ii) KTR10-mutated ichthyosis with confetti (2 cases); (iii) KRT2-mutated superficial epidermolytic ichthyosis (5 cases); and (iv) KRT10-mutated epidermolytic nevus (2 cases). Of note, molecular genetic testing in a third case of extensive epidermolytic nevus revealed a somatic missense mutation (p.Asn186Asp) in the KRT2 gene, detected in DNA from lesional skin at an allelic frequency of 25% and, at very low frequency (1.5%), also in blood. Finally, we report three novel dominant mutations, including a frameshift mutation altering the C-terminal V2 domain of keratin 1 in three familiar cases presenting a mild phenotype. Overall, our findings expand the phenotypic and molecular spectrum of KI and show for the first time that epidermolytic nevus can be due to somatic KRT2 mutation.

The first case of a mosaic superficial epidermolytic ichthyosis diagnosed by Ultra-Deep Sequence.

BACKGROUND: Superficial epidermolytic ichthyosis (SEI), known as ichthyosis bullosa of Siemens (IBS; OMIM No. 146800) before, is a type of keratinopathic ichthyosis due to the KRT2 mutations (NM_000423.3; OMIM No. 600194). Here, we report the first case of SEI caused by a KRT2 mosaic mutation. METHODS: We presented the clinical data of a 5-year-old Chinese boy who suffered from SEI. The histopathological examination and immunofluorescence were performed to rule out immunobullous skin diseases and diseases with subepidermal blisters. Genomic DNA samples were extracted from the lesion tissue and next-generation sequencing was performed. We also confirmed the variant allele frequency (VAF) in different tissues by an Ultra-Deep Sequencing technology. RESULTS: The patient presented with blisters on the lower extremities and linear, superficially hyperkeratotic lesions. Immunofluorescence of IgG, IgA, IgM, C3, C4, and C1q were negative, and the histopathological results showed intraepidermal blisters containing lymphocytes and eosinophils. A heterozygous missense mutation, c.G1459A (p. Glu487Lys), in exon 7 of the KRT2 gene was detected at a 31.17% allele frequency. The same mutation p. Glu487Lys has been described several times in the literature. CONCLUSION: Thus, in our patient, the mosaic mutation explains the blaschkoid ichthyosiform phenotype. To our knowledge, this is the first case of SEI with a KRT2 mosaic mutation.

Gene expression within the periaqueductal gray is linked to vocal behavior and early-onset parkinsonism in Pink1 knockout rats.

BACKGROUND: Parkinson's disease (PD) is a degenerative disease with early-stage pathology hypothesized to manifest in brainstem regions. Vocal deficits, including soft, monotone speech, result in significant clinical and quality of life issues and are present in 90% of PD patients; yet the underlying pathology mediating these significant voice deficits is unknown. The Pink1-/- rat is a valid model of early-onset PD that presents with analogous vocal communication deficits. Previous work shows abnormal α-synuclein protein aggregation in the periaqueductal gray (PAG), a brain region critical and necessary to the modulation of mammalian vocal behavior. In this study, we used high-throughput RNA sequencing to examine gene expression within the PAG of both male and female Pink1-/- rats as compared to age-matched wildtype controls. We used a bioinformatic approach to (1) test the hypothesis that loss of Pink1 in the PAG will influence the differential expression of genes that interact with Pink1, (2) highlight other key genes that relate to this type of Mendelian PD, and (3) catalog molecular targets that may be important for the production of rat vocalizations. RESULTS: Knockout of the Pink1 gene resulted in differentially expressed genes for both male and female rats that also mapped to human PD datasets. Pathway analysis highlighted several significant metabolic pathways. Weighted gene co-expression network analysis (WGCNA) was used to identify gene nodes and their interactions in (A) males, (B) females, and (C) combined-sexes datasets. For each analysis, within the module containing the Pink1 gene, Pink1 itself was the central node with the highest number of interactions with other genes including solute carriers, glutamate metabotropic receptors, and genes associated with protein localization. Strong connections between Pink1 and Krt2 and Hfe were found in both males and female datasets. In females a number of modules were significantly correlated with vocalization traits. CONCLUSIONS: Overall, this work supports the premise that gene expression changes in the PAG may contribute to the vocal deficits observed in this PD rat model. Additionally, this dataset identifies genes that represent new therapeutic targets for PD voice disorders.

Serum lipids, retinoic acid and phenol red differentially regulate expression of keratins K1, K10 and K2 in cultured keratinocytes.

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.

Highly deleterious variations in COX1, CYTB, SCG5, FK2, PRL and PGF genes are the potential adaptation of the immigrated African ostrich population.

Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.

The expression of KRT2 and its effect on melanogenesis in alpaca skins.

In order to investigate the effects of the keratin 2 (KRT2) on alpaca melanocyte in vivo and vitro, the immunohistochemistry (IHC), quantitative real-time PCR (qPCR), Western blot, and alpaca melanocytes transfection methods were used. The results showed that mRNA and protein expression of KRT2 was highly expressed in brown skin in comparison with that in white skin. Moreover, we found that KRT2 was expressed in alpaca melanocytes in vitro by immunocytochemistry. After transfection with KRT2 in alpaca melanocytes, the relative mRNA and protein expression of KRT2, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) in alpaca skin melanocytes was increased with significant differences; a further result was the increase of melanin production. The results suggested that KRT2 functions in alpaca hair color formation, which offered an essential theoretical basis for further exploration of the role of melanogenesis.

Expanding the Clinical and Genetic Spectrum of KRT1, KRT2 and KRT10 Mutations in Keratinopathic Ichthyosis.

Twenty-six families with keratinopathic ichthyoses (epidermolytic ichthyosis, superficial epidermolytic ichthyosis or congenital reticular ichthyosiform erythroderma) were studied. Epidermolytic ichthyosis is caused by mutations in the genes KRT1 or KRT10, mutations in the gene KRT2 lead to superficial epidermolytic ichthyosis, and congenital reticular ichthyosiform erythroderma is caused by frameshift mutations in the genes KRT10 or KRT1, which lead to the phenomenon of revertant mosaicism. In this study mutations were found in KRT1, KRT2 and KRT10, including 8 mutations that are novel pathogenic variants. We report here the first case of a patient with congenital reticular ichthyosiform erythroderma carrying a mutation in KRT10 that does not lead to an arginine-rich reading frame. Novel clinical features found in patients with congenital reticular ichthyosiform erythroderma are described, such as mental retardation, spasticity, facial dysmorphisms, symblepharon and malposition of the 4th toe.

Keratins K2 and K10 are essential for the epidermal integrity of plantar skin.

BACKGROUND: K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. OBJECTIVE: To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. METHODS: Krt2(-/-) Krt10(-/-) mice were generated by crossing Krt2(-/-) and Krt10(-/-) mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2(-/-) Krt10(-/-) mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. RESULTS: K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2(-/-) Krt10(-/-) mice. CONCLUSIONS: This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2(-/-) Krt10(-/-) mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma.

The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.

Loss of keratin K2 expression causes aberrant aggregation of K10, hyperkeratosis, and inflammation.

Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation.

Superficial epidermolytic ichthyosis: a report of two families.

Superficial epidermolytic ichthyosis (SEI), previously known as ichthyosis bullosa of Siemens, is a rare genetic skin condition, characterized by blisters and hyperkeratosis. It can be easily confused with epidermolytic hyperkeratosis, known now as epidermolytic ichthyosis, and genetic testing can be helpful in differentiating between the two conditions. We describe two children with SEI confirmed by genetic testing, including one with a novel mutation. We also describe other affected family members with SEI.

Keratins 2 and 4/13 in reconstituted human skin are reciprocally regulated by retinoids binding to nuclear receptor RARalpha.

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.

Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.

Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.