Post Zygotic, Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer, Producing a Monolateral Palmar Epidermolytic Nevus

Abstract

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

Keywords: EPPK; KIF; NEPPK; genodermatosis; keratin structure; keratins; mosaic.

Figure 1

Patient presentation. (A), picture of both the patient’s hands showing hyperkeratotic lesions only in the right palm. (B,C) pictures of patient’s right palm showing the hyperkeratotic lesions (white arrows), with a nevoid distribution.

Figure 2

HE staining of palmar skin. (A) Control palmar skin. (B) Patient palmar skin. Dashed lines and arrows indicate the joint between nevoid lesion and non or low lesional skin (10× objective). (C) Control palmar skin. (D) Patient palmar skin (20× objective), low lesional skin is on the left side of the dotted line. Bars = 50 μm.

Figure 3

Skin confocal immunofluorescence analysis. Staining was performed using antibodies against K14 (red) and K1 (green). (A) Control palmar skin. (B,C) Patient palmar skin (20× objective). (D) Normal palmar skin (40× objective). (E), Patient palmar skin with K1 and K14 staining merged with DAPI (40× objective, also for (F–I). (F) Capture only showing K1 distribution. (G) Patient skin with merged channels showing K14, K1 and DAPI. (H) Patient skin showing only K1 staining. (I) Patient skin showing only K14 staining. (A–F) Scale bar = 100 µm. (G–I) Scale bar = 50 µm.

Figure 4

Skin confocal immunofluorescence analysis. Staining for p63 (red) and Loricrin (green). (A) Staining with p63 and loricrin merged with DAPI (Blue) of a control section. (B) Staining of a patient section. (C) Patient section (same of B) showing only loricrin signal. (D) Single channel acquisition of the control section showing only loricrin. (E,F) Patient skin stained with loricrin, p63 and DAPI. (A–F) Scale bar = 100 µm, 20× objective. (G–I), staining of patient sections with loricrin, p63 and DAPI at higher magnification; scale bar = 50 µm, 40× objective.

Figure 5

Skin confocal immunofluorescence analysis. Staining for p63 (Green) and K1 (Red). (A) p63 and K1 staining merged with DAPI (Blue) (B) K1 single channel. (C) p63 single channel (A–C, 20× objective). (D) High magnification capture (60× objective) showing nuclear localization of p63 and K1 filament clumping (stars, please see high magnification capture furnished in Figure S1); scale bars = 50 μm. (E) K1 single channel. (F) p63 single channel. Immunofluorescence analysis for p63 and K1 antigens. P63 is identified using Alexa fluor^®488 igG (FITC green fluorescence), laser at 488 nm and 525/50 band pass filter while K1 with Alexa fluor^®568 igG (TRITC red fluorescence), laser at 561 nm and 595/50 band pass filter. Nuclei detection was performed using DAPI (Blu fluorescence), laser at 404 nm and 450/50 band pass filter.

Figure 6

Molecular analysis. (A) Electrophoretic analysis of amplified KRT1 from genomic DNA using to sequence the gene from PBLs DNA extract. (B) Electrophoresis of full length KRT1 cDNA and fragment of exon 1 (right lanes), obtained from the reverse-transcription and amplification using RNA extracted from palmar skin biopsy. Right lanes show specific amplification of exon 1, which are evident by the smaller band due to the presence of the deletion. (C) Electrophoresis of a positive clone containing the deleted K1 exon 1, the vector was digested with BstXI restriction enzyme. (D) Electropherograms showing the sequence of the WT and mutated K1 of the patient obtained from cloned exon1 fragments. (E) Sequence alignment of WT and mutated K1, showing the deleted amino acids. The last 15 residues that belong to the ISIS box (total of 21 residues) are in red.

Figure 7

(A) Model of the K1/K10 dimer [16]. The major domains and the linker regions are identified by different colors. (red, head domain; yellow, coil 1A; cyan, coil 1B; orange, coil 2; blue, tail domain). K10 keratin is represented by gray ribbon and wires. (B) Detail of the head domain of the WT K1 keratin (red). The green color identifies the V1 domain. (C) Detail of the ΔK1/K10 dimer model, characterized by the absence of the V1 domain, used as a starting structure for molecular dynamics simulations.

Figure 8

Time-dependent RMSD calculated for the WT (black line) and ΔK1/K10 (red line) dimers.

Figure 9

Dynamic cross-correlation matrices obtained from PCA analysis of the (A) WT (black line) and (B) ΔK1/K10 (red line) dimers trajectories. Positive correlations are identified by colors ranging from light brown to red, while negative correlations are described by colors ranging from grey to violet.