Malignant transformation of white sponge nevus: a case report of a novel keratin 4 mutation.

BACKGROUND: White Sponge Nevus (WSN) is traditionally considered a benign genetic disorder affecting the oral mucosa, primarily caused by pathogenic mutations in keratin 4 (KRT4) or keratin 13 (KRT13). Despite its benign nature, recent evidence has begun to question the malignant potential of WSN. CASE PRESENTATION: We report a case involving a 70-year-old man who presented with a white lesion on the right floor of his mouth. Initial diagnostic evaluations confirmed the lesion as WSN. Over a one-year follow-up, the lesion underwent malignant transformation, evolving into local epithelial moderate-to-severe dysplasia. Exome sequencing identified a novel insertion mutation in exon 1 of the KRT4 gene, resulting in a deletion-insertion amino acid mutation involving glycine. Single-cell RNA sequencing further revealed altered epithelial proliferation and differentiation dynamics within the lesion. CONCLUSIONS: This case not only expands the known genetic spectrum of KRT4 mutations associated with WSN but also provides preliminary evidence suggesting the malignant potential of WSN. The novel pathogenic mutation in KRT4 is postulated to alter epithelial proliferation and differentiation, thereby raising concerns about the malignant transformation of WSN. Further studies are warranted to confirm these findings.

White Sponge Nevus Caused by Keratin 4 Gene Mutation: A Case Report.

White sponge nevus (WSN) is a rare autosomal dominant disease with a family history, often caused by mutations of the keratin 4 (K4) and keratin 13 (K13) genes in patients. It is characterized by frequently occurred white corrugated folds in the bilateral buccal mucosa with soft texture. On histopathological examination, hyperkeratosis of epithelial cells, edema, and vacuolar changes in the spinous cells are observed in the lesions, despite a normal layer of basal cells. WSN should be differentiated from other oral white spot diseases, mainly oral lichen planus, oral candidiasis, oral white edema, and Heck's disease, to reduce misdiagnosis and unnecessary treatment. At present, there is no specific treatment method. The purpose of this study was to report the clinical data of four WSN patients of the same family with the K4 gene mutation. The occurrence of WSN in a pair of monozygotic twins with very similar clinical presentations was identified for the first time. The gene sequencing results showed that there was a heterozygous deletion (C. 438_440delCAA) in exon 1 of the K4 gene, resulting in an aspartic acid loss in both the proband and his father. Finally, the etiology, pathogenesis, pathological manifestations, clinical manifestations, diagnosis, differential diagnosis, and related treatment methods are discussed to provide a reference for clinical treatment of the disease.

[A boy with white sponge naevus of the buccal mucosa]. / Een jongen met witte afwijkingen op het mondslijmvlies.

White sponge naevus (WSN) is a rare, autosomal dominant disorder that causes various complaints WSN is most commonly found on the buccal mucosa. Clinically, the white, slightly elevated lesions of WSN may be confused with other disorders on oral mucosa. We report a case of WSN in a 14-year-old boy who had complaints for a considerable period of time. WSN is caused by mutations in KRT4 and KRT13.

CD24 and CK4 are upregulated by SIM2, and are predictive biomarkers for chemoradiotherapy and surgery in esophageal cancer.

Definitive chemoradiotherapy (CRT) is a less invasive therapy compared with surgery for some types of cancer; however, the 5­year survival rate of patients with stages II­III esophageal squamous cell carcinoma (ESCC) is only 37%. Therefore, prediction of CRT responders is necessary. Unfortunately, no definitive biomarker exists that is useful to predict survival outcome following CRT. From our previous microarray study, CD24 and keratin 4 (KRT4), which encodes cytokeratin 4 (CK4), were overexpressed in the favorable prognostic epithelial subtype with SIM bHLH transcription factor 2 (SIM2) expression. This study investigated the association between their mRNA and protein expression levels, and clinicopathological characteristics, and also investigated the functions of CD24 in SIM2­mediated tumor differentiation and CRT sensitivity. High CD24 and KRT4 mRNA expression was associated with a favorable prognosis following CRT. Multivariate analyses revealed that high CD24 and CK4 protein expression, as determined by immunohistochemistry, and differentiated type were independent factors for predicting a favorable prognosis in response to CRT. Notably, in cases with low CD24 or CK4, surgery was suggested to be a good therapeutic modality compared with CRT. CD24 and KRT4 were expressed preferentially in differentiated layers of the normal esophageal mucosa, and their mRNA expression in 3D cultured ESCC cells was induced by SIM2 transfection, thus suggesting that CD24 and KRT4 were downstream differentiation markers of SIM2. Furthermore, CD24 small interfering RNA increased the mRNA expression levels of superoxide dismutase 2 and enhanced H2O2 resistance, thus indicating the involvement of CD24 in the radiosensitivity of patients with ESCC; however, it had no effect on cisplatin sensitivity. In conclusion, the two markers CD24 and CK4 may be considered predictive biomarkers for definitive CRT.

Keratin 4 regulates the development of human white sponge nevus.

BACKGROUND: The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). METHODS: Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). RESULTS: The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. CONCLUSIONS: Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

Clinical features and molecular genetic analysis in a Turkish family with oral white sponge nevus.

BACKGROUND: Oral white sponge nevus (WSN) is a rare autosomal dominant benign condition, characterized by asymptomatic spongy white plaques. Mutations in Keratin 4 (KRT4) and 13 (KRT13) have been shown to cause WSN. Familial cases are uncommon due to irregular penetrance. Thus, the aim of the study was: a) to demonstrate the clinical and histopathological features of a three-generation Turkish family with oral WSN b) to determine whether KRT4 or KRT13 gene mutation was the molecular basis of WSN. MATERIAL AND METHODS: Out of twenty members of the family ten were available for assessment. Venous blood samples from six affected and five unaffected members and 48 healthy controls were obtained for genetic mutational analysis. Polymerase chain reaction was used to amplify all exons within KRT4 and KRT13 genes. These products were sequenced and the data was examined for mutations and polymorphisms. RESULTS: Varying presentation and severity of clinical features were observed. Analysis of the KRT13 gene revealed the sequence variant Y118D as the disease-causing mutation. One patient revealed several previously unreported polymorphisms including a novel mutation in exon 1 of the KRT13 gene and a heterozygous deletion in exon 1 of KRT4. This deletion in the KRT4 gene was found to be a common polymorphism reflecting a high allele frequency of 31.25% in the Turkish population. CONCLUSIONS: Oral WSN may manifest variable clinical features. The novel mutation found in the KRT13 gene is believed to add evidence for a mutational hotspot in the mucosal keratins. Molecular genetic analysis is required to establish correct diagnosis and appropriate genetic consultation.

Mutations in the genes for keratin-4 and keratin-13 in Swedish patients with white sponge nevus.

BACKGROUND: White sponge nevus is a rare autosomal dominant disorder that affects the non-keratinised stratified squamous epithelium. Mutations in the genes that encode mucosa-specific keratin-4 and keratin-13 are strongly linked to the manifestation of white sponge nevus. This study involved mutational analysis of the genes encoding keratin-4 and keratin-13 in two Swedish families with white sponge nevus. METHODS: The diagnosis of white sponge nevus was based on disease history, clinical characteristics of the lesions and, in the majority of the cases, histopathological examination. Samples were collected from the affected buccal mucosa using buccal swabs. DNA was subsequently extracted and amplified using touchdown-PCR. The keratin-4 and keratin-13 genes were sequenced, and a genetic analysis was performed. RESULTS: A novel heterozygous missense mutation was identified in exon 1A of the keratin-4 gene in Family 2. In addition, previously reported heterozygous missense mutations were identified in the keratin-4 (E449K, A72V, Q156R, R208H) and keratin-13 (L115P) genes in both families. CONCLUSION: We describe a novel heterozygous missense mutation in the keratin-4 gene of a Swedish family with white sponge nevus. Our results support the notion that mutations in keratin-4 and keratin-13 are the underlying cause of white sponge nevus.

The alcohol-sensitive period during early octavolateral organ development in zebrafish (Danio rerio).

Fetal alcohol exposure can cause Fetal Alcohol Spectrum Disorders (FASD), completely preventable developmental disabilities characterized by permanent birth defects. However, specific gestational timing when developing organs are most sensitive to alcohol exposure is unclear. In this study, we examined the temporal effects of embryonic alcohol exposure on octavolateral organs in zebrafish (Danio rerio), including inner ears and lateral line neuromasts that function in hearing, balance, and hydrodynamic detection, respectively. To determine an alcohol-sensitive period in the first 24 hours post fertilization (hpf), Et(krt4:EGFP)sqet4 zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours. Octavolateral organs of control and alcohol-exposed larvae were examined at 3, 5, and 7 days post fertilization (dpf). Using confocal and light microscopy, we found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at 3 dpf. Only alcohol-exposed larvae from 12-17 hpf had smaller otic vesicles at 5 dpf, smaller saccular otoliths at 7 dpf and fewer saccular hair cells, neuromasts and hair cells per neuromast at 3 dpf. In addition, auditory function was assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. Our results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. This study implies that embryonic alcohol exposure timing during early development can influence severity of hearing deficits. © 2017 Wiley Periodicals, Inc.

Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish.

PURPOSE: To study the feasibility of engineering conjunctival epithelial cell sheets on a temperature-responsive culture dish for ocular surface reconstruction. METHODS: Rabbit conjunctival epithelial cells (rCjECs) were cultured in DMEM/F-12 (1:1) medium. The morphology and phenotype of the rCjECs were confirmed with phalloidin staining, periodic acid-Schiff (PAS) staining, and immunocytochemistry. The rCjECs cultured on a temperature-responsive culture dish for 10 days produced confluent conjunctival epithelial cell sheets. Then, the phenotype, structure, and function of the conjunctival epithelial cell sheets were examined. RESULTS: The conjunctival epithelial cells were compact, uniform, and cobblestone shape. All cultured conjunctival epithelial cells were harvested as intact cell sheets by reducing the culture temperature to 20 °C. Conjunctival epithelial cells were stratified in four to five cell layers similar to the conjunctival epithelium. CCK-8 analysis, 5-bromo-2'-deoxyuridine (BrdU) staining, and the live and dead viability assay confirmed that viable proliferation cells were retained in the cell sheets. Immunohistochemistry for CK4, CK19, and MUC5AC showed the cell sheets still maintained characteristics of the conjunctival epithelium. CONCLUSIONS: A temperature-responsive culture dish enables fabrication of viable conjunctival epithelial cell sheets with goblet cells and proliferative cells. Conjunctival epithelial cell sheets will be promising for reconstruction of the conjunctival epithelium.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

Genes involved in epithelial differentiation and development are differentially expressed in oral and genital lichen planus epithelium compared to normal epithelium.

Lichen planus (LP) is a chronic mucocutaneous disease with unknown cause. Patients with LP often have both oral and genital lesions, but these conditions are often considered as separate diseases and treated accordingly. To find out which genes are differently expressed in mucosal LP compared to normal mucosa and establish whether oral and genital LP are in fact the same disease, whole genome expression analysis was performed on epithelium from 13 patients diagnosed with oral and/or genital LP and normal controls. For confirmation of keratin 4 and corneodesmosin expression, quantitative reverse-transcription PCR and immunohistochemistry were used. Many genes involved in epithelial development and differentiation are differently expressed in epithelium from LP compared to normal epithelium. Several of the differentially expressed genes are common for oral and genital LP and the same biological processes are altered which supports the fact that oral and genital LP are manifestations of the same disease. The change in gene expression indicates that differentiation is altered leading to changes in the epithelial barrier.

Mutation of keratin 4 gene causing white sponge nevus in a Japanese family.

White sponge nevus (WSN) is a rare autosomal dominant disorder characterized by white plaques of oral mucosa; it is benign condition with no effective treatment. The disorder usually manifests during early childhood or adolescence. Mutations of keratin 4 or 13 gene have been identified as causing WSN. The aim of this study is to determine whether keratin 4 or 13 gene mutation was the molecular basis of WSN in a Japanese family. The proband in this family was an 11-year-old boy, with three other people affected by WSN. Genomic DNA was extracted from two affected members and an unaffected member. Segments of keratin 4 and 13 genes were amplified by PCR, and direct DNA sequencing was carried out. Sequence analysis revealed a heterozygous 3bp deletion (N160Del) localized in the helix-initiation motif at the beginning of alpha-helical domain 1A of keratin 4 gene from affected members. One member lacking the phenotype was genetically tested normal. The authors identified a mutation of the keratin 4 gene recurrent in a family affected by WSN. Further investigation of the multifunctional role of keratin genes is warranted in the group of inherited epithelial disorders that may result in identification of effective treatment for this genetic disease.

Fascin and CK4 as biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Several studies have suggested that fascin, cytokeratin 14 and cytokeratin 4 may have significant roles as biomarkers for the progression and survival of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: This study performed immunohistochemistry in tissue microarrays, profiling premalignant lesions and invasive tumors. RESULTS: Fascin increased across the following states as follows: normal-appearing epithelium (26%) to dysplasia (46%) to ESCC (68%), while CK4 was undetectable in ESCC (0%) compared to normal-appearing epithelium (45%) or dysplasia (41%). CK14 was elevated and invariant in expression. In regression analyses, compared to normal-appearing epithelium, higher fascin expression was associated with a 36% increased risk of dysplasia (odds ratio=1.36) and a 56% increased risk of invasive ESCC (odds ratio=1.56). CONCLUSION: Expression of fascin is up-regulated in the transformation from normal-appearing epithelium, through dysplasia, into invasive carcinoma. Expression of CK4, CK14 and fascin did not correlate with patient survival. Fascin has a potential role as an early detection biomarker and CK4 as a tumor marker in ESCC.

Down-regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis.

AIMS: This study aimed to identify relevant keratin subtypes that may associate with the pathogenesis of oral epithelial neoplasms. METHODS AND RESULTS: Expression of all the keratin subtypes was examined by cDNA microarray analysis of 43 oral squamous cell carcinoma (OSCC) cases. Immunohistochemical expression of the major keratins was examined in 100 OSCC and oral epithelial dysplasia (OED) cases. Many changes in keratin expression were observed and, significantly, consistent down-regulation of keratin 4 (K4) and K13 expression was observed. Aberrant expression of K4 and K13 was associated with morphological changes in the affected oral epithelium. Experiments with cell cultures transfected with various keratin subtypes suggested that alterations in keratin subtype expression can cause changes in cell shape and movement. CONCLUSIONS: Aberrant expression of K4 and K13, which are the dominant pair of differentiation-related keratins in oral keratinocytes, indicates dysregulation of epithelial differentiation in OSCC and OED. These keratins, especially K4, may be useful for pathological diagnosis. We propose that the aberrant expression of K4 and K13 and concomitant up-regulation of the other keratins may be one of the causative factors for morphological alterations in the affected epithelium.

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.

The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish.

A tissue-specific transgenic model was employed to test the effects of intron and vector sequences on transgene expression in zebrafish after microinjection. In this model, the 2.3 kb promoter taken from the 5' upstream region of the transcription initiation site of keratin 4 (krt4) was used to drive the enhanced green fluorescence protein (EGFP) reporter gene in a transgenic vector. For assaying the strength of EGFP expression, the effects of including an intron before the EGFP coding region or using different forms of DNA, including circular plasmid, linear full-length plasmid, and the linear transgene coding region without any prokaryotic vector sequence, were tested. After microinjection, the transgene expression was analyzed using transient assays. Consequently, further comparative analysis supported by Fisher's exact test was performed based on the data generated by analyzing the strength of the transgene expression. It was shown that inclusion of an intron in the construct increases the transgene expression in a transient transgenic zebrafish assay. Furthermore, the circular plasmid containing the transgene produced the strongest EGFP expression.

Both all-trans retinoic acid and cytochrome P450 (CYP26) inhibitors affect the expression of vitamin A metabolizing enzymes and retinoid biomarkers in organotypic epidermis.

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.