De Novo Mutation in KRT1 Leads to Epidermolytic Palmoplantar Keratoderma: from Chinese Traditional Treatment to Prenatal Diagnosis Using Whole-Exome Sequencing-Plus.

Congenital skin disorders are a class of complex genetic diseases that are difficult to diagnose and treat. We developed trio whole-exome sequencing-plus (WES-plus) for detecting de novo mutations and evaluated the use of traditional Chinese medicine (TCM) for treating congenital skin disorders. In this study, we successively performed panel-based next-generation sequencing (NGS) and Trio WES-plus in a child with frequent large blisters. Panel-based NGS revealed no pathogenic mutations. Trio WES-plus for resequencing based on cutaneous keratosis of the palms and feet detected a missense mutation (c.1436T>A, p.Ile479Asn) in the coding region of KRT1 in the child but not in his parents. Following prenatal diagnosis, a healthy second baby without the mutation was born. The disease symptoms of epidermolytic palmoplantar keratoderma (EPPK) application were improved by TCM and Western medicine. Our study revealed the pathogenicity of a de novo mutation in human KRT1, which expands the mutation spectrum of EPPK. Trio WES-plus is useful for diagnosing genetic diseases and providing genetic guidance from prenatal diagnosis to treatment.

Proteomic profiling reveals KRT6C as a probable hereterodimer partner for KRT9: New insights into re-classifying epidermolytic palmoplantar keratoderma (EPPK) and a milder form of pachyonychia congenita (PC-K6c) as a group of genetic cutaneous disorders.

Epidermolytic palmoplantar keratoderma (EPPK), a highly penetrant autosomal dominant genodermatosis, is characterized by diffuse keratoses on palmplantar epidermis. The keratin 9 gene (KRT9) is responsible for EPPK. To date, phenotypic therapy is the primary treatment for EPPK. Because KRT9 pairs with a type II keratin-binding partner to function in epidermis, identifying the interaction partner is an essential first step in revealing EPPK pathogenesis and its fundamental treatment. In this study, we proved that keratin 6C (KRT6C) is a probable hereterodimer partner for KRT9. In silico model for KRT6C/KRT9 shows a typical coiled-coil structure in their 2B domains. Proteomics analysis shows that KRT6C/KRT9 pair is in a densely connected protein-protein interaction network, where proteins participate jointly in regulating cytoskeleton organization and keratinization. This study shows that co-immunoprecipitation coupled with mass spectroscopy and proteomics analysis provide a sensitive approach, which compensates for inevitable inadequacies of anti-keratin 6C antibody and helps discover the probable hereterodimer partner KRT6C for KRT9. The acknowledgement of KRT6C/KRT9 pairwise relationship may help re-classify EPPK and PC-K6c (a milder form of pachyonychia congenita, caused by KRT6C) as a group of hereditary defects at a molecular-based level, and lay foundation for deciphering the keratin network contributing to EPPK and PC-K6c. SIGNIFICANCE OF THE STUDY: What is already known about this topic? KRT9 and KRT6C are disease-causing factors for epidermolytic palmoplantar keratoderma (EPPK) and a milder form of pachyonychia congenita (PC-K6c), respectively. EPPK and PC-K6c have some symptom similarities. Keratins are the major structural proteins in epithelial cells. Each of the type I keratin is matched by a particular type II keratin to assemble a coiled-coil heterodimer. The hereterodimer partner for KRT9 is unknown. What does this study add? We discovered and proved that KRT6C is a probable hereterodimer partner for KRT9 in palmplantar epidermis in a native endogenous environment by using co-immunoprecipitation coupled with mass spectroscopy and proteomics analysis, etc. The proteomics analysis shows that KRT6C/KRT9 keratin pair is in a densely connected protein-protein interaction network, where proteins participate jointly in regulating intermediate filament-based cytoskeleton organization and keratinization processes. What are the implications of this work? The new understanding of probable KRT6C/KRT9 pairwise correlation may help re-classify the genetic cutaneous disorders EPPK and PC-K6c as a group of hereditary defects at a molecular-based level, and lay foundation for pathogenic mechanism research in EPPK and PC-K6c. The densely related network components derived from the proteomic data using Metascape in the study and pairwise regulation fashion of specific keratin pairs should attract more attention in the further explorations when investigators concern the physiological functions of keratins and the pathogenesis of related skin diseases.

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.

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.

A de novo mutation of KRT1 in a baby girl causing epidermolytic ichthyosis with impressive epidermolytic palmoplantar keratoderma.

We report a 6-year-old girl showing epidermolytic ichthyosis/epidermolytic hyperkeratosis (EI/EH). Targeted Next Generation Sequencing revealed a de novo, previously unidentified KRT1 mutation. The findings of this study expands the clinical and  spectrum and genotype-phenotype correlation associated with EI/EH.

Annular epidermolytic ichthyosis with palmoplantar keratosis: a unique phenotype associated with interfamilial phenotypic heterogeneity.

BACKGROUND: Annular epidermolytic ichthyosis (AEI) is a rare autosomal dominant ichthyosis that was recently described in 10 separate families in the English literature. There are no reports on the phenotypic heterogeneity of AEI. OBJECTIVES: We investigated, for the first time, a large Chinese AEI pedigree exhibiting interfamilial phenotypic heterogeneity. MATERIALS AND METHODS: We collected clinical data and DNA from the members of the family, and skin lesions were obtained from two patients with different phenotypes. Skin imaging examinations were performed. Whole-exome sequencing (WES) and Sanger sequencing were used to detect gene mutations. RESULTS: The characteristic features of granular layer degeneration in the two biopsies were verified via histological methods. The missense mutation c.1436T > C in KRT1 was detected in all nine patients. CONCLUSION: This study demonstrates that AEI may present with different clinical phenotypes and that mutation analysis for suspected cases is necessary to obtain a precise diagnosis.

Diagnosis and Management of Inherited Palmoplantar Keratodermas.

Inherited monogenic palmoplantar keratodermas are a heterogeneous group of conditions characterised by persistent epidermal thickening of the palmoplantar skin. Palmoplantar keratodermas are grouped depending on the morphology of the keratoderma into diffuse, focal/striate or papular/punctate. Some palmoplantar keratodermas just affect the skin of the palms and soles and others have associated syndromic features which include changes in hair, teeth, nails, hearing loss or cardiomyopathy. Next generation sequencing has helped discover genes involved in many of these conditions and has led to reclassification of some palmoplantar keratodermas. In this review, we discuss the diagnostic features of palmoplantar keratodermas and management options.

Keratin 9 L164P mutation in a Chinese pedigree with epidermolytic palmoplantar keratoderma, cytokeratin analysis, and literature review.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is characterized by hyperkeratotic lesions on palms and soles. The disorder is caused by mutations of keratin 9 (KRT9) or KRT1 gene. METHODS: Epidermolytic palmoplantar keratoderma was diagnosed by physical examination and histopathological analysis in a five-generation Chinese family. Mutation was screened by Sanger sequencing. The palmar expression of multiple cytokeratins were analyzed by tape-stripping and Real-time PCR. Literatures of EPPK with additional symptoms were reviewed. RESULTS: Affected family members showed diffuse palmoplantar keratosis, with knuckle pads, friction-related lesions and a novel additional symptom of palmar constriction. A heterozygous mutation of c.T491C (p.L164P) of KRT9 was found within the helix initiation motif. The hydrophobic effect was decreased and the initiation of coiled-coil conformation was delayed. The KRT16/KRT6 expression were significantly increased in the patients, especially on the right, indicating activation of stress-response and wound-healing cytokeratins. There were also increased KRT9/KRT2, unchanged KRT10/KRT1, and undetectable KRT14/KRT5 expression. The genetic and phenotypic heterogeneity of EPPK with additional symptoms were summarized by literature review. CONCLUSION: The p.L164P mutation of KRT9 caused EPPK with a novel symptom of palmar constriction. The expression of multiple cytokeratins was altered in EPPK patients.

Exome sequencing identifies a KRT9 pathogenic variant in a Chinese pedigree with epidermolytic palmoplantar keratoderma.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is a rare skin disorder and its pathogenesis and inheritability are unknown. OBJECTIVE: To investigate the inheritance and pathogenesis of EPPK. METHODS: Two EPPK cases occurred in a three-generation Chinese family. Patient-parents trio EPPK was carried out and the identified candidate variants were confirmed by Sanger sequencing. RESULTS: A heterozygous missense pathogenic variant, c.488G > A (p.Arg163Gln), in the keratin (KRT) 9 gene was detected in the proband and his son via targeted exome sequencing, and then validated by Sanger sequencing. This pathogenic variant cosegregated with the EPPK in extended family members, and was predicted to be pathogenic by SIFT, PolyPhen2, PROVEAN, and Mutation Taster. This heterozygous variation was not evident in 100 healthy controls. CONCLUSION: This report describes a KRT9 c.488G > A (p.Arg163Gln) variant causing a diffuse phenotype of Chinese EPPK. The current results broaden the spectrum of KRT9 pathogenic variants responsible for EPPK and have important implications for molecular diagnosis, treatment, and genetic counseling for this family.

Identification of a Novel Keratin 9 Missense Mutation in a Chinese Family with Epidermolytic Palmoplantar Keratoderma.

BACKGROUND/AIMS: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis. It is characterized by diffuse yellow keratoses on the palmoplantar epidermis, with an erythematous border. The keratin 9 gene (KRT9) and less frequently the keratin 1 gene (KRT1) are responsible for EPPK. This study aims to identify and analyse genetic defects responsible for EPPK in a Han Chinese pedigree. METHODS: A four-generation Han Chinese pedigree containing five individuals affected with EPPK was recruited. Exome sequencing, Sanger sequencing, and bioinformatics tools were conducted to identify the mutation in this pedigree. HaCaT cells were transfected with either wild-type or mutated KRT9. Confocal laser immunofluorescence assay, imaging processing, and statistical analysis were performed to evaluate wild-type and mutant KRT9 groups. RESULTS: A novel heterozygous c.1369C>T transition (p.Leu457Phe) in exon 6 of the KRT9 gene was identified in four patients. It co-segregated with the disorder in the family. Functional analysis showed that withdrawal of the filament network from the cell periphery and particle formation were present in about 10% of Leu457Phe-transfected HaCaT cells, while approximately 3% of cells transfected with wild-type KRT9 showed this phenotype. The particles in mutant group were larger than that in wild-type group (P-value < 0.05). CONCLUSION: The variant may be the disease-causing missense mutation and produce dominant negative effects by interrupting keratin network formation. This study indicates the pathogenic role of the KRT9 gene mutation in this pedigree with EPPK, and may be helpful in genetic counseling, prenatal diagnosis and gene-targeted therapies of EPPK.

A novel mutation of KRT9 gene in a Chinese Han pedigree with epidermolytic palmoplantar keratoderma.

BACKGROUND: Mutations of keratin 9 (KRT9) gene is a hot research area of epidermolytic palmoplantar keratoderma (EPPK). AIMS: To identify the genes caused the EPPK of a Chinese family. PATIENTS/METHODS: Three cases of lesions were collected for pathological examination. Genomic DNA was extracted from peripheral blood samples of six patients and five healthy individuals and 100 unrelated individuals. Polymerase chain reaction (PCR) was used to amplify exons 1 of KRT9 gene. PCR products were sequenced to identify potential mutations. RESULTS: The lesion pathology of the proband and two ill relatives diagnosed EPPK. A new heterozygous missense mutation (488G>T) was identified in the 488 site of exon 1 of KRT9 gene in all six patients, which resulted in substitution of thymine for guanine, and substitution of leucine acid for arginine acid at position 163 of the KRT9 protein. The same mutation was not found in the five healthy individuals of the family and 100 unrelated individuals. CONCLUSIONS: The new heterozygous missense mutation (488G>T) of KRT9 gene is probably the cause of EPPK in this Chinese family.

Six generations of epidermolytic palmoplantar keratoderma, associated with a KRT9 R163W mutation.

Epidermolytic palmoplantar keratoderma (EPPK) is a rare autosomal dominant skin disorder characterized by diffuse hyperkeratosis on the palms and soles. Whole-exome sequencing (WES) has become a powerful tool for the detection of rare causal variants of Mendelian disorders. However, no causal gene for EPPK in the Uygur population has been identified until now, and no treatment exists than can address the underlying pathology.WES analysis was undertaken on two individuals from a large Uygur EPPK pedigree whose disease locus mapped to 17q21.2 (chr:38994621-39893408) following previous linkage analysis. KRT9 (NM_000226.3:c.487C>T, p.Arg163Trp), and KRT15 (XM_005257346.1:c.212G>T, XP_005257403.1:p.Gly71Val) located in this region, have been identified as two candidate causative genes for EPPK in the Uygur family. Sanger sequencing was conducted on this region in other affected individuals (n = 38) from this family, non-affected individuals (n = 56) from this family and 100 unrelated controls. The missense mutation KRT9 c.487C>T, identified in this large Uygur population, is a potential causative mutation. To date, EPPK has no effective therapy, and siRNA is a potential avenue for EPPK therapy. To investigate this, full-length wild-type Keratin9 (KRT9; pKRT9-WT) and p.Arg163Trp (pKRT9-R163W) were then transfected into HaCaT cells. The small interfering RNAs targeting the KRT9 R163W mutant and wildtype KRT9 were transfected into HaCaT cells, and total RNA isolated at 72 h post-transfection. Quantitative polymerase chain reaction and western blotting were used to analyse the effects of knock-down on KRT9 mRNA and protein levels, respectively. siRNA was shown to specifically inhibit mutant KRT9 mRNA and protein expression (p < 0.01, with 95% confidence limits). Our study suggests that KRT9 is a causal gene for EPPK. This information is helpful for understanding the pathogenesis of EPPK in the Uygur population and raises the possibility of designing a novel siRNA treatment strategy for this population of EPPK patients.

A STAT1-gain-of-function mutation causing Th17 deficiency with chronic mucocutaneous candidiasis, psoriasiform hyperkeratosis and dermatophytosis.

During recent years, inborn errors of human IL-17 immunity have been demonstrated to underlie primary immunodeficiencies with chronic mucocutaneous candidiasis (CMC). Various defects in receptors responsible for sensing of Candida albicans or downstream signalling to IL-17 may lead to susceptibility to Candida infection. While CMC is common in patients with profound T cell immunodeficiencies, CMC is also recognised as part of other immunodeficiencies in syndromic CMC, or as relatively isolated CMC disease. We describe a 40-year-old woman with a clinical picture involving cutaneous bacterial abscesses, chronic oral candidiasis and extensive dermatophytic infection of the feet. By whole exome sequencing, we identified a STAT1-gain-of-function mutation. Moreover, the patient's peripheral blood mononuclear cells displayed severely impaired Th17 responses. The patient was treated with antifungals and prophylactic antibiotics, which led to resolution of the infection. We discuss the current knowledge within the field of Th17 deficiency and the pathogenesis and treatment of CMC.

Possible anticipation in familial epidermolytic palmoplantar keratoderma with the p.R163W mutation of Keratin 9.

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease characterized by diffuse hyperkeratosis of the epidermis of the palm and sole with an erythematous margin. The Keratin 9 (KRT9) and Keratin 1 genes are responsible for EPPK. Several previous studies have focused on the genetic basis of EPPK; however, genetic anticipation has not yet been reported. We described a four-generation family with EPPK and identified a p.R163W mutation of KRT9. We observed a decrease in the age of onset in three consecutive generations in the family of the proband, indicating possible genetic anticipation in this familial EPPK. Further studies are needed to elucidate the mechanisms of anticipation in EPPK.