ORIGINAL ARTICLE

Mutations Affecting 10 Surface-Exposed Residues Highlight the Structural Basis of Phenotypic Variation in Epidermolytic Haris Mirza1, Anil Kumar2, Brittany G. Craiglow1, Jing Zhou1, Corey Saraceni1, Richard Torbeck3, Bruce Ragsdale4, Paul Rehder5, Annamari Ranki6 and Keith A. Choate1,7

Epidermolytic ichthyosis (EI) due to KRT10 mutations is a rare, typically autosomal dominant, disorder characterized by generalized erythema and cutaneous blistering at birth followed by hyperkeratosis and less frequent blistering later in life. We identified two KRT10 mutations p.Q434del and p.R441P in subjects presenting with a mild EI phenotype. Both occur within the mutational “hot spot” of the keratin 10 (K10) 2B rod domain, adjacent to severe EI-associated mutations. p.Q434del and p.R441P formed collapsed K10 fibers rather than aggregates characteristic of severe EI KRT10 mutations such as p.R156C. Upon differentiation, from p.Q434del showed significantly lower apoptosis (P-valueo0.01) compared with p.R156C as assessed by the TUNEL assay. Conversely, the mitotic index of the p.Q434del epidermis was significantly higher compared with that of p.R156C (P-valueo0.01) as estimated by the Ki67 assay. Structural basis of EI phenotype variation was investigated by homology-based modeling of wild-type and mutant K1–K10 dimers. Both mild EI mutations were found to affect the surface-exposed residues of the K10 alpha helix coiled-coil and caused localized disorganization of the K1–K10 heterodimer. In contrast, adjacent severe EI mutations disrupt key intermolecular dimer interactions. Our findings provide structural insights into phenotypic variation in EI due to KRT10 mutations. Journal of Investigative Dermatology (2015) 135, 3041–3050; doi:10.1038/jid.2015.284; published online 6 August 2015

INTRODUCTION sub-divided into 1A, 1B, 2A, and 2B domains (Coulombe and Keratin intermediate filaments are heteropolymers of at least Omary, 2002). Short amino acid sequences at the N- and one type 1 and one type 2 keratin and are the primary C-terminus of the rod domain are hotspots for pathogenic stress-bearing structures in epithelial tissues (Fuchs and mutations in epidermal . Such mutations in KRT1 and Cleveland, 1998; Szeverenyi et al., 2008). (K1) KRT10 cause a heterogeneous group of disorders classified as and K10 are exclusively expressed in post-mitotic suprabasal epidermolytic ichthyosis (EI; DiGiovanna and Bale, 1994), keratinocytes, whereas K5 and K14 are limited to the whereas mutations affecting corresponding regions of KRT5 proliferative basal epidermal compartment. All keratin pro- and KRT14 result in the epidermolysis bullosa simplex (EBS; teins have a highly conserved, central α-helical rod domain Lee et al., 2012). and variable head and tail regions. The α-helix is further EI patients present with blistering and erythema early in life. With age, the frequency of blisters decreases, and the skin 1Department of Dermatology, School of Medicine, Yale University, New Haven, becomes hyperkeratotic, although the severity and distribution Connecticut, USA; 2Department of Biology and Chemistry, Laboratory of of hyperkeratosis can vary (DiGiovanna and Bale, 1994). The Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland; 3St Luke’s-Roosevelt Hospital—Mount Sinai Health System, New York, New York, majority of EI cases are caused by autosomal dominant USA; 4Central Coast Pathology Laboratory, San Louis, California, USA; 5The missense mutations (Szeverenyi et al., 2008), and to our Dermatology Medical Group of Oxnard and Camarillo, Camarillo, California, knowledge there are no reports of KRT10 singleaminoacidin- 6 USA; Department of Dermatology and Allergology, University of Helsinki and KRT10 Skin and Allergy Hospital, Helsinki University Central Hospital (HUCH), frame deletion mutations. Here we present two Helsinki, Finland and 7Department of Genetics and Pathology, School of mutations, one missense and the other an in-frame tri- Medicine, Yale University, New Haven, Connecticut, USA nucleotide deletion, both presenting with a mild EI phenotype. Correspondence: Keith A. Choate, Department of Dermatology, School of Apart from architectural support, keratins have a crucial Medicine, Yale University, New Haven, Connecticut, USA. role in differentiation, growth, and proliferation E-mail: [email protected] (Santos et al., 2002; Paramio et al., 2007). Disruption of these Abbreviations: EBS, epidermolysis bullosa simplex; EI, epidermolytic ichthyosis; K1, keratin 1; SE, surface exposed; WT, wild-type non-structural functions due to mislocalization (Choate et al., Received 13 February 2015; revised 12 May 2015; accepted 26 May 2015; 2010) or gain of potentially toxic functions by aggregation accepted article preview online 15 July 2015; published online 6 August 2015 (Li and Törmä, 2013) may determine clinical outcomes of

© 2015 The Society for Investigative Dermatology www.jidonline.org 3041 H Mirza et al. Structural Basis of Phenotype Variation in EI

specific keratin mutations. Rod domain mutations found in Detection of mutations in 2B domain of K10 severe EI show accumulation of cytotoxic keratin aggregates Sequencing of KRT10 in K101 and K102 identified identical, in suprabasal keratinocytes, epidermolysis, disrupted de novo, heterozygous trinucleotide, in-frame deletions epidermal differentiation, and basal cell hyperproliferation c.1301_1303delAAC (Supplementary Figure S2A online), (Fuchs et al., 1994). In contrast, mild EI exhibits limited leading to loss of a highly conserved glutamine residue in filament disruption, absence of cytotoxic aggregates, and the 2B rod domain (p.Q434del; Supplementary Figure S3 formation of perinuclear tonofilaments (Syder et al., 1994). online). In K103, a c.G1322C mutation (Supplementary Similarly, frameshift mutations affecting the tail domain Figure S2B online) resulted in substitution of a conserved of K10 found in ichthyosis with confetti cause disruption arginine residue (PhyloP score of 1.387; Supplementary of epidermal differentiation and hyperproliferation, without Figure S3 online) with proline (K10 p.R441P). Both of these cytolysis or keratin aggregation. Instead, filaments mislocalize mutations, to our knowledge, are previously unreported, and to the nucleolus (Choate et al., 2010). Here we investigate neither of them has been found in the 1000 genomes or the the pathobiology of intermediate filament reorganization in exome variants sever control data sets. two KRT10 mutations, p.Q434del and p.R441P, identified in individuals with mild EI. Disruption of keratin network without epidermolysis in mild EI To understand the reasons for filament collapse rather than epidermis cytotoxic aggregation in mild EI keratinocytes, we sought to Histopathological features of mild (K101) versus severe EI study the K1–K10 polymer structure in wild-type (WT) and (K104) epidermis correlated with phenotype. The basal mutant states. However, such investigations were limited by epidermis in both cases appeared healthy, whereas the the absence of a K1–K10 crystal structure (Sung et al., 2013). cytolysis and vacuolization appeared in the lower most Capitalizing on the homology between keratin rod domains, spinous layer in severe EI (p.R156C) and progressed with we generated a computational model of K1–K10 using the further differentiation (Figure 1f and i). Mild EI epidermis K5–K14 crystal structure as a template (Lee et al., 2012). This (p.Q434del) showed limited signs of cellular degeneration in model predicted multiple inter- and intra-molecular interac- first few layers of spinous cells without frank epidermolysis tions in the rod domain of the K1–K10 dimer crucial for (Figure 1e and h). Both mild (Figure 1e and h) and severe EI filament organization. Using this approach, we investigated epidermis (Figure 1f and i) showed acanthosis, hyperkeratosis, the impact of EI-associated mutations on these interactions and hypergranulosis. Histopathological findings comparable and correlated them with observed phenotypes. to those of our p.Q434del case have also been reported for K10 p.R441P (Niemi et al., 1990). RESULTS AND DISCUSSION Expression of K10 was reduced (Figure 2a, Supplementary Case descriptions Figure S4A online) with expansion of K5 and K14 expression fi We identi ed three unrelated kindreds presenting with a mild to suprabasal keratinocytes along with increased KRT5 and fi EI phenotype. The rst case (K101) was an 11-year-old female KRT14 transcription in p.Q434del and p.R156C epidermis who reported no skin abnormalities at birth and developed (Figure 2c and d, Supplementary Figure S4B online). K10 progressive scaling in early childhood. On examination, filaments in mild EI (p.Q434del) exhibited perinuclear generalized mild and locally more severe hyperkeratosis was collapse without the aggregates seen in severe EI seen on the neck, back, elbows, hands, knees, lower legs, and (Figure 2a). Similar perinuclear filament collapse was also – feet (Figure 1a c). There was slight palmoplantar keratoderma reported in K10 p.R441P epidermis (Niemi et al., 1990). In without a history of blistering or skin fragility. The second case addition, a collapse and a decreased expression of K10 (K102) was a 32-year-old female who had apparently normal binding partner K1 was also observed (Figure 2b, skin at birth but developed scaly patches on her knees, Supplementary Figure S4A online). As histopathological – elbows, and dorsal hands around 3 4 months of age, which features of K10 p.Q434del are nearly identical with other eventually progressed to generalized scaling with areas of reports of mild EI, (Niemi et al., 1990; Syder et al., 1994) we corrugated hyperkeratosis by age 2. Recent examination focused on K10 p.Q434del mutant for further pathobiological fi revealed generalized ne white scales and areas of hyperker- comparisons with the classical severe EI case K10 p.R156C. fi atosis and licheni cation most notable over the knees, dorsal Transfection of WT KRT10 into basal keratinocytes resulted hands, ankles, and feet, without palmoplantar keratoderma in the formation of a well-arborized filament network – (Supplementary Figure S1A E online). She was otherwise (Figure 3a). In contrast, p.R156C mutant K10 generated healthy and reported no family history of skin fragility or punctate clumps without filament formation and was blisters. In both kindreds, the mutation appears de novo. The found to disrupt the native keratin network (Figure 3a). third subject (K103) and her family members were previously p.Q434del mutant K10 expression on the other hand reported as Curth-Macklin resembling EI generated a collapsed filament network instead of aggre- (Niemi et al., 1990; Bonifas et al., 1993). This subject gates (Figure 3a). presented with thick, corrugated hyperkeratosis over joints (Supplementary Figure S1F–J online). We compared patholo- Epidermal homeostasis in mild EI epidermis gical features of our index case with subject K104 (K10 p. R156C), a well characterized KRT10 mutation found in severe Lower rate of apoptosis in mild EI keratinocytes.Tofurther EI (Syder et al., 1994). study mutant K10 cytotoxicity, we isolated primary

3042 Journal of Investigative Dermatology (2015), Volume 135 H Mirza et al. Structural Basis of Phenotype Variation in EI

Wild type K10 p.Q434del K10 p.R156C

* **

Figure 1. Clinical and histopathological features of mild epidermolytic ichthyosis (EI). (a) Mild EI subject (K101; p.Q434del) shows hyperpigmentation of the upper back with notable fine white scaling and hyperkeratosis around elbows. Similar scaling and hyperkeratosis was also observed around knees and shins (b), as well as ankles and feet (c). (d) and (g) show histology of the wild-type skin. Panels (e) and (h) exhibit histological features of mild EI, highlighting epidermal acanthosis, hyperkeratosis, and hypergranulosis*, with limited cellular degeneration in the uppermost layer of the spinous layer. Panels (f) and (i) show classical EI epidermis of K10 p.R156C, highlighting cytolysis** beginning from the first suprabasal cells layer. Cytolysis was remarkably absent in the suprabasal layer of mild EI epidermis (e and h). Black bars = 100 μm.

keratinocytes from healthy controls, p.Q434del, and p.R156C gain of cytotoxic function in p.R156C cells such as keratin subjects. Monolayers were grown to confluence and calcium aggregation, rather than cell fragility owing to a mere absence of switch performed to induce KRT10 expression. A negligible functional keratin filaments (Figure 3a). A pro-cell survival function number of healthy and p.Q434del keratinocytes exhibited of K10 has been proposed (Chen et al., 2006); however, overt cell pyknosis (Supplementary Figure S5 online). In contrast, signifi- death was not observed in a K10 knockout mice (Reichelt and cantly higher number of cells (P-valueso0.01) in differentiating p. Magin, 2002). Altogether, the increased rate of shear-independent R156C keratinocytes became pyknotic (Supplementary Figure S5 apoptosis observed here (Figure 3b and c, Supplementary Figure S5 online), and cells sloughed off from the substrate without online) appears to be a gain of cytotoxic function by EI-associated application of mechanical stress (Figure 3b). KRT10 mutations. More importantly, the higher apoptotic rate in To identify the mechanism of cytotoxicity in EI cell lines, we p.R156C cells compared with mild EI cells (Figure 3c, Supple- analyzed the mutant and WT primary keratinocytes for signs mentary Figure S5 online), along with the absence of epidermolysis of apoptosis using the TUNEL assay (Figure 3c). We induced the in p.Q434del skin (Figure 1e and h), highlights the importance ++ cells to express K10 by Ca -mediated differentiation. K10 p.R156C of cell death in determining EI severity. Altogether, our findings cells showed significantly higher fraction of apoptotic cells support the hypothesis that keratin aggregation is responsible for (P-values 0.01) at 56.3% of total, compared with 29.3% in p. keratinocyte cell death, that widespread cytolysis causes severe Q434del and 17.1% in WT keratinocytes (Figure 3c). In the absence symptoms in EI patients, and that the collapsed filaments of mild EI of mechanical stress, the increased apoptosis likely resulted from the are well tolerated by the keratinocytes.

www.jidonline.org 3043 H Mirza et al. Structural Basis of Phenotype Variation in EI

K10 K1 Wild type K10 p.Q434del K10 p.R156C

K5 K14 K6 Inv Wild type K10 p.Q434del K10 p.R156C

Figure 2. Alterations of epidermal intermediate filament network in mild epidermolytic ichthyosis (EI). (a and b) Paraffin-embedded sections of wild-type tissue showed pancytoplasmic keratin 1 (K1) and K10 filaments. Filament retraction and perinuclear K1 and K10 accentuations were observed in p.Q434del, whereas p. R156C showed suprabasal intermediate filament (IF) clumping. (c and d) Compared with wild type, both EI epidermises showed expansion of anti-K14 and -K5 staining to the suprabasal layer, along with suprabasal expression of K6 (e). (f) Expansion of anti-involucrin staining was also observed in both mild and severe EI. Yellow bar = 100 μm, green and red bars = 350 μm. Dotted lines represent the dermal–epidermal junction.

Increased mitotic index in mild EI epidermis. Involucrin staining also observed in both p.Q434del and p.R156C suprabasal cells was expanded in both mild and severe EI epidermis (Figure 2f), (Figure 2e). Basal cell hyperproliferation response to epidermal hypergranulosis was present (Figure 1e, h, f and i), and differen- stress (Wraight et al., 2000) is a feature of both mild and severe tiation-associated expression of epidermal keratins was disrupted EI (Porter et al., 1998). Interestingly, a higher mitotic index was (Figure 2a–e), suggesting an abnormal epidermal differentiation observed in the p.Q434del epidermis compared with the pR156C program. Expression of K6 in the spinous layer, a hallmark of tissue, despite a lower apoptotic rate (Figure 3b–d, Supple- epidermal hyperproliferative disorders (Ramírez et al., 1998), was mentary Figure S5A and B online). Similar hyperproliferation was

3044 Journal of Investigative Dermatology (2015), Volume 135 H Mirza et al. Structural Basis of Phenotype Variation in EI

also reported in the K10 knockout mice even in the absence Structural basis of phenotype variability in EI of epidermolysis or cell fragility (Reichelt and Magin, 2002). Taken together, these findings suggest the presence Homology-based modeling predicts inter- and intra-molecular of a pro-proliferation signal in EI epidermis, independent of interactions in K1–K10 2B domain dimer. As phenotype epidermolysis. variation in EI appears to be a function of two distinct keratin

DAPI K10 K5 Wild type K10 p.Q434del

20 μm K10 p.R156C

DAPI TUNEL K14,Ki67 Wild type Wild type A 300 82.4% B 17.1%

200

100 Wild type

0 0 200 400 600 800 1K

K10 p.Q434del K10 p.Q434del A 69.9% B 120 29.3% 90

60

Cell counts 30 K10 p.Q434del 0 0 200 400 600 800 1K K10 p.R156C K10 p.R156C 150 A 45.4% B 53.6% 100

50 K10 p.R156C 200 µm 100 μm 0 0 200 400 600 800 1K FL1-A

Figure 3. Apoptosis and proliferation in mild and severe epidermolytic ichthyosis (EI) epidermises. (a) HaCaT cells expressing either wild-type (WT) or p.Q434del or p.R156C mutant (K10). Monolayers were co-stained with anti-K5 and anti-K10. WT co-localizes with K5, whereas p.R156C formed aggregates and disrupted the K5 filaments. p.Q434del formed perinuclear filaments (b) Confluent monolayers of keratinocyte from WT, p.Q434del, and p.R156C subjects were differentiated followed by DAPI staining. More than 50% of the p.R156C cells showed pyknosis, whereas WT and p.Q434del cells showed limited cell death. (c) Differentiating p.QR156C primary keratinocytes showed significantly higher TUNEL-positive cells compared with WT and p.R156C cells (P-valueso0.01). + + (d) Epidermises stained with anti-Ki67 and anti-K14. p.Q434del epidermis showed significantly higher K14 Ki67 cells compared with wild-type and p.R156C epidermises (P-valueo0.01).

www.jidonline.org 3045 H Mirza et al. Structural Basis of Phenotype Variation in EI

K10 α-Helix (rod domain)

Head 2 Tail 1A 1B 2B A

K10

K1

E396 Y400 E415 Y482 E478 3.6Å R369

3.8Å 2.5Å

3.3Å 2.8Å E434 K443 R450

Heptad units

400 460 Human | K10

433 493 Human | K1

Inter- intra-chain interactions α-Helical organization Hydrophobic interactions Salt bridges (heptads g & e) Hydrogen bonds Hydrophobic interactions (heptads a & d) Electrostatic interactions Surface (heptads b, c & f) Interaction with H2O

R441P Y449D Y449C E445K Q434del L452P K10 R422E

K1 L453P K439E Q447P Severe EI R450P L435P I446T Mild EI L442Q

Figure 4. Mapping of molecular interactions and keratin 10 (K10) mutations on the K1–K10 2B domain dimer model. (a) Graphical representation of K10 domains with green bars highlighting K10 mutational “hotspots” and red bar represents the portion of K10 α-helix modeled. K1–K10 dimer model highlighting the intermolecular interactions (magenta) between K1 (blue) and K10 (red) residues. (b) Coiled-coil heptad repeat assignments and inter-/intra-chain interactions in K1–K10 dimer. Details of these interactions are summarized in Supplementary Table 1 online (c) Mapping of K10 mutations on the K1–K10 2B dimer with mild mutations in green and severe mutations in red. K10 p.Y449 residue mutation (yellow) results in severe phenotype in mutation K10 p.Y449D or mild phenotype in K10 p.Y449C. Details on interaction and specific mutations are summarized in Table 1.

filament morphologies (i.e., cytotoxic keratin aggregates of severe K5–K14 dimer including interactions of K10–E415 and K1–K443, EI and collapsed filaments of mild EI), we compared the structural K10–R450 and K1–E478, as well as hydrogen bond interactions consequences of mild EI mutations in our subjects with between K10–Y400 and K1–E434, and K10–R450 and K1–Y482 previously reported severe EI mutations located in the proximity. (Figure 4a and b, Supplementary Table S1 online). Sequence and We generated computational models of WT and mutant K1–K10 structure alignments highlighted that the C-terminal “trigger- dimers (Figure 4, Supplementary Figure S6A online, Table 1 and motif” region with a high density of hydrophobic interactions in Supplementary Table S1 online). Visualization of the WT model K1–K10 is similar to the K5–K14 crystal structure (Figure 4a and showed conservation of non-polar interactions with respect to b). Structural superimposition of the K10–K1 dimer model onto

3046 Journal of Investigative Dermatology (2015), Volume 135 H Mirza et al. Structural Basis of Phenotype Variation in EI

Table 1. Epidermolytic ichthyosis mutations in the K1–K10 2B coiled-coil K10 mutations Corresponding K14 mutations

Amino Acid substitution/deletion EI phenotype Interactions Q-mean values1 Amino acid substitution/deletion EBS phenotype

K10-p.R422E2 Mild SB 1.19 K14-p.R388C3 EBS-WC K14-p.R388H3 EBS-WC K14-p.R388G3 EBS-K K14-p.R388P3 EBS-WC/-K K10-p.Q434del4 Mild SE 0.59 — K10-p.L435P5 Severe K1–Y463, K1–L468, K1–M464 0.99 K14-L401P3 EBS-WC K10-p.K439E2 Mild6 K10–E443 1.32 —— K10-p.R441P4 Mild SE, K10–E445 0.75 —— K10-p.L442Q2 Severe K1–I479 1.34 K14-L408M3 EBS-WC K10-p.E445K2 Severe K10–R441, K1–R483 1.33 K14-E411K3 EBS-DM K14-E411K3 EBS-K K14-E411del3 EBS-WC K10-p.I446T2 Mild — 1.36 K14-I412F3 EBS-WC K14-I412N3 EBS-DM K10-p.Q447P2 Mild SE, K10–N444 1.00 K14-A413P3 EBS-WC K14-A413T3 EBS-K K10-p.Y449D2 Severe HTM, K1–Y482, K1–I479, K1–L486 1.38 K14-Y415H3 EBS-DM K14-Y415H3 EBS-K K10-p.Y449C2 Mild HTM, K1–Y482, K1–I479, K1–L486 1.35 K14-Y415C3 EBS-WC K14-Y415C3 EBS-K K10-p.R450P2 Severe K1–E478 (salt bridge) 1.13 K14-R416P3 EBS-DM K1–Y482 (H-bond) K10-p.L452P2 Severe SB 1.03 K14-p.L418V3 EBS-K K10-p.L453P2 Severe HTM, K1–L486 1.02 K14-p.L419Q3 EBS-DM Abbreviations: EBS-DM, epidermolysis bullosa simplex—Dowling − Meara type; EBS-K, epidermolysis bullosa simplex—Koebner type; EBS-WC, epidermolysis bullosa simplex—Weber − Cockayne type; EI, epidermolytic ichthyosis; SB, salt bridge; SE, surface exposed. 1Q-mean values of the homology-based models of mutant and wild-type K1–K10 dimer. 2EI-associated K10 sequence variants found in interfil database (Szeverenyi et al., 2008). 3EBS-associated K14 sequence variants found in interfil database (Szeverenyi et al., 2008). 4Mutations presented in this paper. 5Unpublished mutations (presented at SID2014 by Wang et al., 2014). 6Mild EI phenotype with limited keratin filament disruption.

K14–K5 structure showed that K10 Cys401 shares an identical Keratin rod domains adapt a coiled-coil configuration due to a conformational position as the K14 Cys367 (Lee et al., 2012) repeated pattern (heptad repeat) of charged and hydrophobic amino and mediates a disulphide bond between independent K10–K1 acid residues designated as abcdefg.Inanα-helical secondary struc- dimers (Supplementary Figure S6A online), supporting the ture, hydrophobic residues (a or d) are buried, whereas salt-bridge validity of our models. residues, assigned e or g, further stabilize this secondary structure. Next, we mapped the disease-causing missense or deletion Together they are classified as interface residues. This configuration mutations onto the K1–K10 dimer. Our subjects and previously leaves the charged surface-exposed (SE) residues (b, c or f) in direct reported cases were classified as mild EI if they had late onset and/ contact with water-filled cytoplasm (Hanukoglu and Fuchs, 1983). or localized distribution of skin lesions, whereas cases with the Disruption of the interface residues could have serious consequences onset of symptoms at birth and generalized skin disease were for filament formation as they provide the thermodynamic driving classified as severe EI (Figure 4c, Table 1). Similar to K14 2B force for oligomerization. Conversely, SE residue alterations are better domain mutations (Lee et al.,2012;Table1),K10EImuta- tolerated (Lee et al., 2012). On the basis of the heptad repeat tions segregated between “trigger-motif” residue mutations and assignments of the mutated residues, we segregated K10 mutations mutations proximal to it (Figure 4c, Supplementary Figure S6 as affecting either interface (salt bridge and hydrophobic) or SE online, Table 1). residues (Figure 4b, Table 1, Supplementary Figure S7 online).

www.jidonline.org 3047 H Mirza et al. Structural Basis of Phenotype Variation in EI

K1–K10 WT Q433 D437 K10

Y432 L435 Y465 L468 K1 M469

K1-K10 p.Q434del Q433 D437 (K101–K102) K10 Y432 L435 Y465 L468

K1 M469

K1–K10 WT R441 3.4Å E445 K10

K1

K1–K10 p.R441P P441 (K103) E445 K10

K1

Figure 5. 2B domain dimer models of surface-exposed residue keratin 10 (K10) mutations (a) K10 p.Q434del (K101 and K102) and K1 wild-type dimer model. The model predicted loss of hydrophobic intermolecular interactions between adjoining K10–L435 with K1 coil residues Y465, L468, and M469. (b) K10 p.R441P (K103) and K1 wild-type dimer model. This model predicted loss of an intra-molecular interaction between R441 and E445 residues of K10, leaving the remaining inter- and intra-molecular interactions intact. The crucial intermolecular interactions (magenta) described in Figure 3a remain unaffected by p.Q434del and p. R441P mutations.

KRT10 SE residue mutations present with mild EI phenotype. maintained. Consequently, resulting filaments are stable, albeit Both mutations in our mild EI subjects affect SE residues collapsed (Figures 2a and 3a). Similar to K10 p.Q434del, deletion (Figures 4 and 5, Table 1, Supplementary Figure S7 online). of an SE residue in K14, K14 p375del also presents with mild EBS The proline substitution in K10 p.R441P (Figure 5b) disrupts a (Table 1; Supplementary Figures S7 and S9 online; Chen et al., minor intra-molecular interaction between K10–R441 and K10– 1993). Altogether, deletion or substitution of SE residues in type 1 E445, whereas the neighboring dimer interactions remain intact epidermal keratins is usually well tolerated and present with mild (Figure 5b, Table 1, Supplementary Table S1 online). Likewise, phenotypes, with the exception of mutations involving the highly the missense K10 p.Q447P mutation also affects an SE residue in conserved “helix termination motif” (HTM; Figure 4, Table 1). the 2B domain (Figure 4b and c, Table 1, Supplementary Figures These mutations include K5 p.T469P, K5 p.E475K, K5 p.E475G, S7 online; Sheth et al., 2007). As both of these mutations confer a K1 p.T481P, and K14 p.R417P (Supplementary Figures S7 and mild EI phenotype, such mutations may be better tolerated. S6B online). Severe phenotypes of the SE mutations in HTM Furthermore, KRT14 mutations at the sites corresponding to K10– could be due to disruption of additional tetramer interactions. For R447, K14 p.A413P (Natsuga et al., 2011), and K14 p.A413T example, K14 p.R417P mutation disrupts a crucial tetramer (Chao et al., 2002) also present with mild EBS phenotypes interaction between K14 R417 and K5 Q454 of two separate (Table 1; Supplementary Figure S7 online), as do other SE muta- dimers (Supplementary Figure S6B online). Another reason could tions––i.e., K14 p.T319P (García et al., 2011) and K5 p.G476D be the presence of a dense array of interactions in HTM (Abu Sa’d et al., 2006). conserved between K5–K14 and K1–K10 dimers (Lee et al., The deleted K10 p.Q434 codon in our mild EI cases K101 and 2012; Figure 4b, Supplementary Table S1 online). No HTM-SE K102 (Figure 4c, Table 1, Supplementary Figure S7 online) is also mutations have been described in KRT10 thus far. an SE residue that is not directly involved in inter- or intra- molecular interactions (Figures 4 and 5a, Supplementary Figure Disruption of dimer interactions determines disease severity in S7 online, Table 1). A model of the mutated K1–K10 dimer interface mutations. On the basis of our models, interface (Figure 5a) suggests that the deletion of K10–Q434 leads to the mutations that do not alter K1–K10 dimer interactions and/or do loss of three simultaneous hydrophobic interactions between not cause major helix disruptions present with mild EI (Figure 4, adjoining K10–L435 with K1 residues Y465, L468, and M469 Table 1, Supplementary Table S1, Supplementary Figure S7 (Figure 5a); however, strong intermolecular polar interactions are online). For example K10 p.R422E is a polar–polar conservative

3048 Journal of Investigative Dermatology (2015), Volume 135 H Mirza et al. Structural Basis of Phenotype Variation in EI

substitution of a salt-bridge residue, which results in mild disease MATERIALS AND METHODS (Figure 4b and c, Table 1; Supplementary Figures S7 and S8 Human subjects online). Mild EBS in mutation on the corresponding residue of Written informed consent from K102, K103, and K104 and parental K14 R388 supports this observation (Abu Sa’d et al., 2006; permission along with child assent for K101 were obtained. This Bolling et al., 2011; Table 1; Supplementary Figures S7 and S8 study was approved by the Yale Human Investigational Committee – online). Similarly, hydrophobic residue mutations K10 I446T and and complies with the Declaration of Helsinki Principles. Subjects – K10 K439E present with milder phenotype as affected residues provided punch biopsies and blood samples. Patient biopsies were do not contribute to any additional interactions (Figure 4b and c, taken from the lateral side of upper-third of their thigh. Supplementary Figure S7 online, Table 1). – Conversely, interface mutations that disrupt K1 K10 dimer Keratinocyte culture interactions present with severe phenotypes as seen in the cases A measure of 3 mm punch biopsies were transported in DMEM with of K10 p.E445K, p.R450P, and p.L452P SB residue mutations 2 × penicillin/streptomycin (Invitrogen, Waltham, MA). Biopsies (Figure 4b and c, Table 1, Supplementary Figure S6 online; were incubated in 1 × dispase overnight for dermal–epidermal Szeverenyi et al., 2008). Corresponding KRT14 mutations, K14 p. separation. Keratinocytes were recovered from the epidermal sheet E411K and K14 p.R416P (Wood et al., 2003; Glász-Bóna et al., using 0.05% trypsin and plated onto the feeder layer of mitomycin C– 2009; Table 1, Supplementary Figure S7 online), also present treated 3T3 cells in the DMEM/F12 medium. Established keratinocyte with severe EBS. Hydrophobic interface residue mutations such ++ as K10 p.L435P, (Syder et al., 1994), p.L442Q (Chipev et al., colonies were split and expanded in low Ca medium. For in-vitro fl 1994), p.Y449D (Makino et al., 2012), and p.L453P (Virtanen differentiation of primary lines, cells were grown to con uence on et al., 2001) impact dimer interactions and consequently (Figure tissue culture glass slides (Falcon, Franklin Lakes, NJ). Confluent ++ 4b and c, Table 1, Supplementary Figure S7 online) also confer a keratinocytes were switched to a high Ca medium and cultured for more severe phenotype. an additional 72 hours to induce differentiation (Xie et al., 2005). Not all mutations in corresponding residues between K10 and K14 behave similarly: K10 p.L452P, another SB mutation, Homology-based structural modeling of K1–K10 dimer presents with severe EI phenotype (Figure 4b and c, Table 1, The model for the WT K1–K10, as well as K10 Q434del-K1 and K10 Supplementary Figure S7 online), whereas its equivalent K14 R441P-K1 mutant dimers were generated using Swiss-model automated mutation (p.L418V) showed a mild EBS phenotype (Rugg et al., model generation server (http://swissmodel.expasy.org). Template 2007; Table 1; Supplementary Figure S7 online). This could be search was made using Blast and HHBlits against the updated SWISS- “ due to non-conservative substitution of lysine with a helix MODEL template library. Templates were selected on the basis of the ” breaking proline residue in K10 p.L452P in addition to the highest percentage sequence similarity. Models were built based on the disruption of critical salt-bridge interaction, whereas the milder target-template alignment using Promod-II and modeler (Biasini et al., disease phenotype in corresponding K14 p.L418V could be 2014). Coordinates that were conserved between the target and the attributed to non-polar conservative substitution, (Table 1; template were translated from the template to the model. Insertions and Supplementary Figures S7 and S8 online). deletions were remodeled using a fragment library followed by side To our knowledge, structural analysis of EI-associated muta- tions using homology-based modeling has been previously chain rebuilding. The geometry of the resulting model was regularized fi fi fi unreported. Similar approach has been used for genotype– using a force eld. The nal model quality was assessed and quanti ed phenotype correlation in EBS (Liovic et al., 2001; Natsuga et al., by QMEAN for the estimation of the global and per-residue local model 2011); however, these models ignore unique inter- and intra- quality. The figures are drawn using pymol and chimera. Coiled-coil molecular interactions between keratins, leaving genotype– heptad repeat assignments of 2B domain residues were calculated using phenotype correlation of several EBS mutations unexplained. http://embnet.vital-it.ch/software/COILS_form.html. As the 2B rod domain is highly conserved across keratins, our genotype–phenotype correlation method may predict disease severity of other keratin mutations. CONFLICT OF INTEREST fl We have presented two KRT10 mutations K10 p.Q434del and The authors state no con ict of interest. K10 p.R441P associated with mild EI phenotype. Our findings suggest that diversity of clinical presentation in EI is a function of ACKNOWLEDGMENTS We thank Young Lim and Jonathan L Levinsohn for critical review of the two distinct ultrastructural keratin morphologies in suprabasal manuscript and Hu Rong-Hua for technical assistance. This work was keratinocytes––i.e., collapsed keratin filaments in mild disease supported by Doris Duke Charitable Foundation Clinical Scientist Develop- and keratin aggregation in severe disease. We confirmed the higher ment Award to KAC. HM was supported by James Hudson Brown-Alexander cytotoxicity of keratin aggregates by observing the increased rate of Brown Coxe Postdoctoral Fellowship in the Medical Sciences. keratinocyte apoptosis independent of mechanical stress, in cells expressing severe EI p.R156C mutant K10 compared with the cells SUPPLEMENTARY MATERIAL expressing mild EI mutant K10 p.Q434del. Furthermore, using Supplementary material is linked to the online version of the paper at http:// homology-based modeling, we illustrated that mild EI KRT10 www.nature.com/jid mutations either affect SE residues (as seen in our subjects) or – interface residues without altering K1 K10 intermolecular interac- REFERENCES tions essential for keratin polymerization, whereas severe EI results ’ – Abu Sa d J, Indelman M, Pfendner E et al. (2006) Molecular epidemiology of from mutations affecting residues essential for K1 K10 interactions, hereditary epidermolysis bullosa in a Middle Eastern population. J Investig hence the deposition of cytotoxic keratin aggregates. Dermatol Symp Proc 126:777–81

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3050 Journal of Investigative Dermatology (2015), Volume 135