Keratin 13 mutations associated with oral white sponge nevus in two Chinese families

Abstract

White sponge nevus (WSN) is an autosomal dominant hereditary disease. Keratin 4 (KRT4) and Keratin 13 (KRT13) gene mutations were involved in the WSN. We recruited two WSN Chinese families, and oral lesion biopsy with hematoxylin and eosin staining showed that patients had significant pathological characteristics. The mutations of KRT4 and KRT13 gene were detected by PCR and direct sequencing. The multiple alignments of KRT13 from 23 diverse species homology analyses were performed by the ClustalW program. The KRT13 expression was measured by Real-Time RT-PCR and Western blot analysis. Sequencing analysis revealed two mutations of KRT13 gene: one mutation was 332T>C and amino acid change was Leu111Pro. Another mutation was 340C>T and amino acid change was Arg114Cys. The sequence of KRT13 was highly conserved. Real-Time RT-PCR and Western blot analysis results show that KRT13 expression level is lower in patient but keep almost no change in mRNA level. When cells were treated with MG132, KRT13 protein level was increased and kept almost the same in normal and patient cells. We identified two heritable mutations in the KRT13 gene, which were associated with the development of WSN. The abnormal degradation of KRT13 protein of WSN may probably associate with the abnormal ubiquitination process.

Keywords: Gene mutation; Keratin 13 (KRT13); Oral disease; Ubiquitination; White sponge nevus (WSN).

Fig. 1

Two WSN pedigrees from China and clinicopathological report. (A) Pedigree of proband's family from Hunan Province, China (WSN-A). There are 28 cases in this family. Arrow indicates proband A, the 43-year-old male. (B) Pedigree of proband's family from Zhejiang Province, China (WSN-B). Arrow indicates proband B, the 29-year-old male. (C) White spongy plaques on the tongue, the buccal mucosa of normal, proband A from WSN-A family and proband B from WSN-B family. (D) Histopathology of the proband A lesion from WSN-A family. Thickened epidermis and transparent cytoplasm in keratinocytes, the epithelium is generally thickened, showing both hyper parakeratosis and acanthosis with the intact basal layer expression. Hematoxylin–eosin for all straining, original magnification 100 × or 400 ×. (a) Normal epidermis (100 ×). (b) Epithelial hyperplasia, parakeratosis, acanthosis (100 ×). (c) Stratum basale has 1–2 layers of cells, similar to the normal epidermis (400 ×). (d) The up arrow shows an infiltrate of inflammatory cells, the down arrow indicates that the small vessels of infiltrates have extravasated blood (400 ×). (e) The up arrow shows that spinous cells are loosening, the down arrow indicates intracellular edema with a characteristic basket-weave appearance (400 ×). (f) The up arrow shows vacuolization of the spinous cells, the down arrow shows that spinous cells have cytoplasmic basophilic mucoid secretions (400 ×).

Fig. 2

Two KRT13 mutations in two Chinese families with WSN. (A) Partial DNA sequences of exon 1A of the KRT13 gene from the WSN-A family. The arrow indicates the position of the mutation 332T>C, the mutation predicts the amino acid change L111P in the KRT13 polypeptide from the WSN patient. (B) Partial DNA sequences of exon 1A of the KRT13 gene from the WSN-B family. The arrow indicates the position of the mutation 340C>T. The mutation predicts the amino acid change R114C in the KRT13 polypeptide. (C) The exon 1A of the KRT13 gene and the reported mutations being discovered till now. (D) A portion of the amino acid sequence of KRT13 is shown from 23 diverse species. Alignments of amino acid sequences for human, chimpanzee, northern white-cheeked gibbon, olive baboon, gorilla, black-capped chickadee, common marmoset, cow, sheep, horse, elephant, dog, northern greater galago, rabbit, rat, mouse, pig, Tasmanian devil, cat, guinea pig, platypus, xenopus and zebrafish KRT13 proteins. Red means published associated mutations in KRT13 gene; “*” means that the residues or nucleotides in that column are identical in all sequences in the alignment; “:” means that conserved substitutions have been observed; “.” means that semi-conserved substitutions are observed.

Fig. 3

Protein expression of KRT13 is decreased in WSN patient cells. (A) Observation of oral mucosal epithelial cell morphology from normal and WSN patient. (B) The growth curve of WSN patient cell line and normal control cell line (P < 0.05). (C) Quantification of KRT mRNA (P < 0.05). All images shown are representative and data are mean ± s.e.m. of n ≥ 3 independent experimental groups. Results are representative of three independent experiments. All experiments were performed at least three times with similar results. (D) Analysis of the protein expression between normal and WSN. (E) Analysis of the protein expression in oral mucosal epithelial cells with/without MG132 treatment.