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2386 East Heritage Way, Suite B, Salt Lake City, Utah 84109 USA Phone +1-877-628-7300 • Email—[email protected] www.pachyonychia.org Archives of Biochemistry and Biophysics 508 (2011) 123–137

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Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

Review Keratin gene mutations in disorders of human skin and its appendages

Jean Christopher Chamcheu a, Imtiaz A. Siddiqui a, Deeba N. Syed a, Vaqar M. Adhami a, Mirjana Liovic b,c, ⇑ Hasan Mukhtar a, a Department of Dermatology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA b Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Slovenia c National Institute of Chemistry, Ljubljana, Slovenia article info abstract

Article history: Keratins, the major structural protein of all epithelia are a diverse group of cytoskeletal scaffolding pro- Available online 19 December 2010 teins that form intermediate filament networks, providing structural support to keratinocytes that main- tain the integrity of the skin. Expression of keratin genes is usually regulated by differentiation of the Keywords: epidermal cells within the stratifying squamous epithelium. Amongst the 54 known functional keratin Epidermal keratin genes in humans, about 22 different genes including, the cornea, hair and hair follicle-specific keratins Intermediate filaments have been implicated in a wide range of hereditary diseases. The exact phenotype of each disease usually Keratin gene mutation reflects the spatial expression level and the types of mutated keratin genes, the location of the mutations Keratinopathic genodermatoses and their consequences at sub-cellular levels as well as other epigenetic and/or environmental factors. Skin fragility Epidermolysis bullosa The identification of specific pathogenic mutations in keratin disorders formed the basis of our under- Epidermolytic ichthyosis standing that led to re-classification, improved diagnosis with prognostic implications, prenatal testing Pachyonychia congenita and genetic counseling in severe keratin genodermatoses. Molecular defects in cutaneous keratin genes Gene and pharmacologic therapies encoding for keratin intermediate filaments (KIFs) causes keratinocytes and tissue-specific fragility, Disease models accounting for a large number of genetic disorders in human skin and its appendages. These diseases are characterized by keratinocytes fragility (cytolysis), intra-epidermal blistering, hyperkeratosis, and keratin filament aggregation in severely affected tissues. Examples include epidermolysis bullosa simplex (EBS; K5, K14), keratinopathic ichthyosis (KPI; K1, K2, K10) i.e. epidermolytic ichthyosis (EI; K1, K10) and ichthyosis bullosa of Siemens (IBS; K2), pachyonychia congenita (PC; K6a, K6b, K16, K17), epidermolytic palmo-plantar keratoderma (EPPK; K9, (K1)), monilethrix (K81, K83, K86), ectodermal dysplasia (ED; K85) and steatocystoma multiplex. These keratins also have been identified to have roles in apoptosis, cell proliferation, wound healing, tissue polarity and remodeling. This review summarizes and discusses the clinical, ultrastructural, molecular genetics and biochemical characteristics of a broad spectrum of keratin-related genodermatoses, with special clinical emphasis on EBS, EI and PC. We also highlight cur- rent and emerging model tools for prognostic future therapies. Hopefully, disease modeling and in-depth understanding of the molecular pathogenesis of the diseases may lead to the development of novel ther- apies for several hereditary cutaneous diseases. Ó 2010 Elsevier Inc. All rights reserved.

Structure and function of the skin a multilayered, non-vascularized, stratified squamous epidermal tissue overlying the thick fibrous connective dermal tissue, which The human skin is the largest organ of the body, being strategi- then sits on the hypodermis (Fig. 1a). The multilayered epidermal cally located at the interface between the interior and exterior, it sheet comprises four (five in palmoplantar skin) distinct cell layers. provides the first line of defense against external insults. Histolog- The basal layer contains the partially characterized epidermal ically, the skin reveals a built-in synchrony in the outer most epi- keratinocyte stem cell population, which divide and are committed dermis, delineates the existence of a tremendous degree of to terminal differentiation as they move up, forming the suprabasal coordination between tissue-cell layers, and culminates to mediate and upper layers of the epidermis. The thicker spinous (suprabasal) a multiplicity of functions. From outside-in the skin is composed of layers overlay the basal layer, and as cells move up upon commit- ment to terminal differentiation, they gradually become flattened as they enter the granular layer where they collapse and com- ⇑ Corresponding author. Address: Department of Dermatology, University of Wisconsin, Madison, Medical Sciences Center, #B-25, 1300 University Avenue, mence nuclear and organelle degradation and active lipid and pro- Madison, WI 53706, USA. Fax: +1 608 263 5223. tein secretion. This protein and lipid secretion together with cell E-mail address: [email protected] (H. Mukhtar). flattening is critical in maintaining the barrier function of the

0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2010.12.019 124 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137

Fig. 1. Skin structure and keratin intermediate filament in triton extracted cultured keratinocyte cytoskeleton. (a) Cross section of the human skin tissue showing the three distinct skin components. (b) Triton X-100 extracted keratinocyte cytoskeleton in culture stained with keratin 14 antibody (LL002; green filaments) shows a dense network of keratin intermediate filament bundles. The protein scaffold linked to its associated complexes forms the main resilience structure of the epithelial keratinocytes. (Reproduced from [133] with permission from the publisher.) uppermost, cornified layer that are constantly desquamated for tis- forms a network of 10–12 nm wide, and represents about three- sue homeostasis while providing the barrier potential. It is clearly quarters of all known IF in humans. KIFs build into a dense, three- demonstrated that mutations in many cutaneous tissue associated dimensional transcellular and highly dynamic cytoskeleton network keratin genes are responsible for a variety of genetic skin diseases, structure spanning within the nucleus and extending to the cell characterized by compromised specific cell-tissue integrity, periphery, where they anchor and interact with cell–cell (desmo- impairing the ability of the skin to form a proper barrier and with- somes) and cell–matrix (hemidesmosomes) adhesion complexes stand constant physical insults [1,2]. (Fig. 1b) [3,4]. This organization provides structural stability, flexibil- ity, and ensures the mechanical integrity of the different epithelial cells and tissues. The keratins vary in size between 40–70 kDa and Epithelial keratin biology are divided into two groups based on molecular weight: the smaller or low molecular weight acidic type I (40–64 kDa, with PI: 4.7–6.1) Intermediate filaments (IFs) and the keratin IF (KIFs) and the larger or high molecular weight neutral-basic type II (52– 70 kDa, with PI: 5.4–8.4) subgroups of IF proteins [5]. The newly The cell cytoskeleton of all multi-cellular organisms consists of established consensus nomenclature for mammalian keratin genes three abundant filament systems, which play important roles in and proteins is grouped into three categories: (1) epithelial keratins, the organization and mechanical integrity of tissue cells: the actin (2) hair keratins and (3) keratin pseudogenes [6,7]. This nomencla- microfilaments (MFs; 7–10 nm diameter), intermediate filaments ture now includes 28 type I (KRT9, KRT10, KRT12–KRT20, KRT23– (IFs; 10–12 nm diameter), and interconnected microtubules KRT28, KRT31–KRT40) genes coding for (K9, K10, K12–K20, K23– (MTs; 25 nm diameter). Each filament system is built from a family K28, K31–K40) and 26 type II (KRT1-KRT8; KRT71-KRT86) genes cod- of proteins with cell-tissue specific regulation of expression, with ing for (K1-K8; K71-K86) keratins. In the human genome, the genes each protein family being encoded by the corresponding gene fam- encoding type I and type II keratins are mainly clustered at two dif- ily. IFs are by far the most complex of the cytoskeletal proteins ferent loci on chromosomal regions 17q12-q21 and 12q11-q13, with at least 60 different IF proteins subcategorized into six broad respectively. The epidermal type I keratin genes e.g. KRT1, KRT2 types based on tissue-specific expression, sequence similarity and and KRT5, each comprise 9 exons, whereas the genes coding for epi- protein structure (for details see www.interfil.org). dermal Type II keratins e.g. KRT10 and KRT14, each consist of 8 exons 1 The KIFs are the most abundant structural IF protein constitu- (see www.interfil.org and [8]). ents of keratinocytes which form the epidermis. KIFs are encoded by large, well conserved multigene family coding for proteins that Structural and assembly properties of KIFs Similar to all IFs, keratins share a head-rod-tail structural do- 1 Abbreviations used: KIFs, keratin intermediate filaments; IFs, intermediate main organization, with the basic polypeptide structure consisting filaments; HIP, helix initiation motif; HTP, helix termination peptides; EB, epider- of a central a-helical coiled-coil rod domain of about 310 amino molysis bullosa; BMZ, basement membrane zone; EBS, epidermolysis bullosa simplex; NFJS, Naegeli–Franceschetti–Jadassohn syndrome; DPR, dermatopathia acids in size. This central rod domain consist of four helical seg- pigmentosa reticularis; DDD, Dowling-Degos disease; PTC, premature termination ments (1A, 1B, 2A and 2B) that are interrupted by three short codon; KPI, keratinopathic ichthyosis; BCIE, bullous congenital ichthyosiform eryth- non-helical flexible linker regions (L1, L12 and L2), and are flanked roderma; EHK, epidermolytic hyperkeratosis; IHCM, ichthyosis hystrix of Curth- by variable, non-helical amino-terminal head and carboxy-termi- Macklin; SPPK, striate palmo-plantar keratoderma; IBS, ichthyosis bullosa of Siemens nal tail domains (Fig. 2a). The rod domain consists of a repeated se- (IBS); SEI, superficial epidermolytic ichthyosis; PPK, palmo-plantar keratoderma; PFB, pseudofolliculitis barbae; PC, pachyonychia congenita; IPCRR, International PC quence of seven amino acid residues (a-b-c-d-e-f-g)n known as Research Registry; ED, ectodermal dysplasia. ‘‘heptad repeats’’. The positions ‘‘a’’ and ‘‘d’’ are known to be occu- J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 125

ger and more extensible than their type I counterpart [12]. How- ever, these end domains interacts with other filaments and their associated linkers, possibly serving as substrates for post-transla- tional modifications, and to regulate structural organization and functional properties [13,14]. The filamentous stage of keratins consist of the heteropolymeric pairing of one type I and one type II keratin molecule. The heterodimers are formed by coiled-coil association of their corresponding rod domains with both partici- pating monomers exhibiting an in-register parallel alignment. The heterodimers align laterally in an overlapping staggered and antiparallel fashion, forming tetramers which then polymerize to elongated chains and form KIFs via lateral stacking [15] (Fig. 2b).

General features of hereditary cutaneous keratin diseases

In addition to providing structural function, it is evident from human disorders and mouse genetics that the keratin cytoskeleton has more complex roles, which have been associated with addi- tional five broadly defined functions: regulation of apoptosis, cell architecture, stress response, protein synthesis and organelle and vesicle (re)distribution functions [16]. They form complex signal- ing networks, interacting with various kinases, adaptor and apop- totic proteins to regulate cell growth and cell cycle progression [17,18]. They likely regulate apoptosis via various mechanisms [19], namely by acting as a phosphate ‘‘sink’’ for stress-activated kinases, and preventing the activation of pro-apoptotic substrates Fig. 2. Molecular structure and assembly of keratin intermediate filaments (KIF). (a) [20] or by the regulation of key effectors of the stress-induced met- Schematic representation of type I and type II keratin polypeptide domain abolic responses through phosphorylation of specific epitopes on structural organization. Of the 54 different human keratin genes, each keratin keratins [17,21]. Additionally, during physiological wound healing, molecule consists of a central alpha-helical rod domain which is composed of four helical segments, 1A, 1B, 2A and 2B that are interrupted by three flexible non- keratins may regulate various signaling pathways resulting in the helical linker domains L1, L12 and L2. The rod domain begins and ends with highly regulation of protein synthesis and cell size. conserved sequence motifs, helix initiation (HIP) and helix termination (HTP) Tremendous progress in our understanding of the etiology and peptides and is flanked by head and tail domains, respectively. (b) Keratin molecular pathogenesis of cutaneous keratins has formed the basis intermediate filaments assembly; Keratin polymerization obligatorily begins with the formation of coiled-coil obligate heterodimer structures involving winding for re-classification, improved diagnosis and pre-implantation around each other of the central rod domains of type I and type II polypeptides, a genetics with prognostic implications. Moreover, the discovery of requirement underlying the pair wise transcriptional regulation of keratin genes specific mutations has facilitated genetic counseling and prenatal in vivo. The heterodimers then associate (side-by-side) and assemble in an testing for severely affected families [22,23]. overlapping staggered and antiparallel fashion to form stable tetramers. Tetramers then associate end-to-end to form protofilaments and finally, four protofibrils laterally build keratin intermediate filaments. Each filament contains approxi- mately eight protofilaments wound around each other in a rope-like structure, Heritable keratin-related cutaneous diseases forming the 10–12 nm wide KIF network. (Reproduced from [133] with permission from the publisher.) Several human diseases are caused by defects in genes which encode IF proteins. In the epidermis and associated skin append- ages, pathogenic mutations in the coding sequence of keratins pied by hydrophobic residues, considered to be critical for coiled- and their associated linker proteins have been discerned as the coil formation. In addition, near the middle of the 2B domain is a molecular basis of a vast majority of cutaneous disorders. These discontinuity in the heptad repeat (helix inversion), forming the wide ranges of abnormal genetic skin, appendages and membrane ‘‘stutter’’ region, a highly conserved segment among IFs. This re- fragility pathologies are commonly termed genodermatoses. These gion is suggested to play specific roles in the elongation and rota- include about 22 different keratin genes, as well as cornea, hair and tional characteristics of keratins, but does not take part in the hair follicle-specific keratins (see the IF database www.interfil.org coiled-coil heterodimer formation [9]. and [8]). Two decades ago, it was shown for the first time that EB At the beginning of the 1A and the end of the 2B rod domains, simplex was the first human genetic skin blistering disease shown are the highly conserved helix initiation motif (HIP) and helix ter- to be caused by dominant-negative mutations in genes encoding mination peptides (HTP), respectively, which consist of 20 amino an IF proteins, i.e. the basal epidermal keratins K5 and K14 [24– acid sequences in the different keratin gene families. These motifs 26]. Since then, our understanding of several other genodermatos- are critical during the overlapping interactions of KIF assembly, es due to defects in keratin genes has considerably advanced. In explaining why mutations in these sequences interfere with the most conditions, the associated pathology results from fragile early stages of filament elongation [10,11]. Because of this, the he- keratinocytes expressing the mutated keratin. These diseases com- lix boundary motifs are mutation ‘‘hot spots’’ in a wide range of monly termed keratinopathic genodermatoses are individually inherited keratin-related diseases. The terminal head domain re- rare (typically, less than 1:25–50,000 live births), but can be devas- gions consist of the subdomains variable (V1) and homologous tating and incapacitating to affected individuals, incurably affect- (H1) and the terminal tail subdomains H2 and V2, and the end ing their quality of life and being occasionally lethal in severe (E) domains regions (Fig. 2). Subtle differences in the head and tail episodes. For most of these disorders, there exists a good correla- domains mostly account for the diversity between and within the tion between the type of mutated keratin gene, the nature and po- individual keratin group, which results in type II keratins being lar- sition of the mutation in the polypeptide, the extent to which the 126 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 mutation alters the properties of keratin assembly, and the severity ease pathology and genotype–phenotype correlations within a of the clinical phenotype. broad spectrum of keratin disorders. In EBS, two major subtypes Over 90% of pathogenic mutations in keratinopathies are mis- have been defined: suprabasal and basal EBS [29]. Within the scope sense mutations with a small number of small in-frame insertion of this review, only keratin-related will be described. The supraba- vs deletion mutations and a few intronic splice site defects leading sal EBS types such as lethal acantholytic EB (mutations in desmo- to larger in-frame deletions. At the protein level, the consequences plakin), plakophilin deficiency (caused by plakophilin-1 of mutant polypeptides are the expression at normal or near-nor- mutation) and the basal EBS subtypes; EBS caused by mutations mal levels with substitutions, deletions or insertion of a different in plectin (EBS with muscular dystrophy and EBS Ogna), mutations amino acid. The heterodimers of the protein formed from one mu- in plectin and a6b4 integrin (EBS with pyloric atresia) beyond the tant protein and the wild-type keratin partner then integrate into scope of this review (for theses subtypes see www.interfil.org and the keratin network rendering the cytoskeleton susceptible to col- [23]). Clinical EBS is usually present at birth and is characterized by lapse upon mild or no physical stress [27,28]. Additionally, genetic intra-epidermal blistering within the basal layer of the epidermis testing for mutations in KIF and their associated linker proteins, and often with involvement of mucosal epithelia. Blistering which and their site specific expression, reflecting the phenotypic expres- tends to heal without scarring is often associated with mechanical sion pattern delineated various subtypes and variants of each ker- stress, and is usually caused by mutations in keratin K5 or K14. The atin-related disorders. Examples include, epidermolysis bullosa pathogenic mutations usually occur within regions of the keratin simplex (K5, K14), keratinopathic ichthyosis (K1, K2, K10), pal- genes that encode ‘‘hotspots’’ in the protein structure, namely mo-plantar keratoderma (K9), pachyonychia congenita (PC) the H1 domain of the head region (only for type II keratins), two including PC-6a, PC-6b, PC-16 and PC-17 (K6a, K6b, K16 and segments (1A and 2B) of the rod domain, and the central linker re- K17), and monilethrix (K81, K83 and K86), etc. (see www.inter- gion L12 [8]. Upon mild physical trauma, the keratin filament net- fil.org; www.pachyonychia.org and [6,7,12,23]). work is easily compromised, resulting in structural failure of the affected keratinocytes and loss of tissue integrity (reviewed in [12,30]). The degree of severity of the clinical phenotype has been Epidermolysis bullosa (EB) directly linked to the position of the pathogenic mutation along the domains of the polypeptide backbone. Recent evidence provides EB is a large and heterogeneous group of genetically defined some exceptions to the latter, with milder disease phenotypes mechano-bullous skin fragility disorders characterized by fluid resulting from pathogenic mutations in the conserved hot spot re- filled blistering or erosion of the skin and mucous membrane gion of the KRT genes [31,32]. Based on the clinical severity, recent occurring in response to mild or no physical trauma. About 1 in re-classification distinguishes four major EBS subgroups: (a) the 20,000 individuals are affected by any of the several EB types rec- generalized Dowling-Meara EBS (EBS-DM; OMIM 131760), (b) ognized currently. Electron microscopy and immunofluorescence other generalized non-DM EBS (gen non-DM EBS; OMIM antigen mapping have been fundamental in understanding these 131900), (c) the localized EBS (EBS-Loc; OMIM 131800) and (d) genodermatoses and revised classification distinguished four ma- EBS with mottled pigmentation (EBS-MP; OMIM 131960) [29]. jor subtypes. Based on the level of skin cleavage within the cutane- In both generalized forms, the most severe Dowling-Meara sub- ous basement membrane zone (BMZ) [29], we now distinguish type and the milder generalized non-Dowling-Meara subtype, also between: (i) the intra-epidermal EB simplex with cytolysis and previously known as the Koebner form, affected individuals pres- fluid filled blister formed intra-epidermally within the basal kerat- ent generalized and pronounced blistering at birth, while the local- inocytes, usually caused by mutations in either keratin 5 or keratin ized EBS is milder with blistering confined to palmar and plantar 14 gene, (ii) the intra-lamina lucida, junctional EB, with the split regions of the body. Nevertheless, other not yet identified genetic occurring at the level of the lamina lucida and resulting in blister- or epigenetic modifiers and environmental factors, such as patient ing with no obvious structural abnormality of tonofilaments, lifestyle and climate condition, clearly influence the phenotypic which is caused by defects in laminin-332, collagen XVII, or a6b4 expression as different subtypes of EBS have been associated with integrin, (iii) the sub-lamina densa, dystrophic EB, with cleavage the same mutation in several instances [33–35]. However, other at the superficial dermis, underneath the lamina densa at the level additional factors are suggested to exacerbate disease severity of the anchoring fibrils (which link the epidermis and dermis), [36,37]. caused by mutations in the gene that encodes collagen VII, often The generalized EBS-DM subtype is the most severe form being with documented abnormality in tonofilaments, and (iv) the mixed manifested at birth with erythema, widespread blistering, erosions type Kindler syndrome. The intra-epidermal EBS is further sepa- and areas of denuded skin presenting spontaneous clusters of blis- rated into two subgroups, the basal and suprabasal types, respec- ters also called ‘‘herpetiform’’ at multiple sites of the body which tively including newly described types such as those due to improve with age (Fig. 3a). Progressive palmo-plantar keratoderma desmoplakin or plakophilin mutations [29]. becomes the chief complaint in adulthood. Other hallmarks in- To date, all EB subtypes have been characterized at the ultra- clude callosite formation (Fig. 3b), secondary bacterial infections structural and molecular levels, with more than 1000 mutations al- and sepsis, involvement of mucous membranes, nail dystrophy, ready described in more than 21 genes encoding for structural ker- healing of lesions without scarring and involvement of the oral atins in the human skin, its appendages and the mucous mucosa. Inflammation of blisters may be preceded by the forma- membranes. Amongst these includes about 193 KRT5 and KRT14, tion of transient milia, and healing of the skin with hypo- and 115 KRT1 and KRT10 and 67 PC mutations (www.interfil.org; hyperpigmentation [27]. Diagnostic criteria include immuno epi- www.hgmd.cf.ac.uk and [8]). tope antigen mapping and ultrastructural examination of the skin, often present a characteristic pathognomonic electron dense Epidermolysis bullosa simplex (EBS) – diseases of K5/K14 mutations aggregates of K5 and K14 proteins in the cytoplasm of basal kerat- inocytes harboring the mutation [38]. Epidermolysis bullosa simplex (EBS) is a group of rare predom- The pathogenic mutations in EBS-DM are usually missense inantly autosomal dominant genetic skin diseases affecting mutations residing within the highly conserved helix boundary approximately 1:25,000–50,000 live births of the population motifs (the HIP of the 1A segment and the HTP of the 2B segment). [27,29]. EBS is the first identified and best studied variant of kera- More than one-third of the reported EBS-DM cases are caused by tin disorders and has become the prototype for understanding dis- the KRT14 gene ‘‘hot spot’’ mutation in a highly conserved arginine J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 127

Fig. 3. Clinical features of epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI). (a) A child’s hand with severe generalized Dowling-Meara form of EBS, characterized by widespread herpetiform blister that heals without scar formation. (b) An Adult with severe EBS-DM whose feet present with painful plantar callosities. (c) Epidermolytic ichthyosis patient showing sharp massive hyperkeratosis of the lower back and (d) a diffuse hyperkeratosis of the hand and flexures with an erythromatous background. (Reproduced from [133] with permission from the publisher.) residue (Arg125) in the HIP of K14. The likely reason for this genet- ton are far less severe than those seen in EBS-DM and some ic hotspot may be owing to the conserved hypermutable CpG dinu- cases of gen-non-DM EBS. Therefore, based on the site of the cleotide known in all type I keratins, which upon mutation leads to pathogenic mutation in keratins expressed by keratinocytes of the substitution of the arginine codon (CGC) for cysteine (TGC) or the basal epidermal layer, it is for the most cases possible to in- histidine (CAC) with unique phenotype. fer a disease phenotype [40,41]. The gen-non-DM EBS is a more moderate subtype characterized at birth or in early infancy by generalized non-clustered blistering. Autosomal recessive epidermolysis bullosa simplex, EBS-AR The clinical presentation in majority of the cases is moderate, with- out any extra-cutaneous involvement, but with palms, soles and Although EBS is mostly inherited in an autosomal dominant extremities being mostly affected, usually in response to minor mode, approximately 5% of EBS cases are inherited in a recessive trauma and often induced by increased ambient temperature. mode [42], with about 10 different KRT14 mutations) including The disease-associated mutations in the gen non-DM EBS are more nonsense, missense, splice site, deletion and deletion vs insertion centrally located in the rod domain and sometimes more widely mutations identified in (EBS-AR) cases [43]. Yusukawa et al., also distributed along both KRT5 and KRT14 genes, involving the non- reported cases of recessive EBS with compound heterozygous helical linker segments (reviewed in [12]). mutations in K5 [9]. The EBS-loc, a clinically mild phenotype and frequent form previously known as EBS-Weber Cockayne (EBS-WC), is charac- Naegeli–Franceschetti–Jadassohn syndrome (NFJS) and terized by late appearing skin blistering restricted to areas of dermatopathia pigmentosa reticularis (DPR) greater friction or trauma such as hands and feet. Children are not affected until they start to crawl or walk and blister forma- Naegeli–Franceschetti–Jadassohn syndrome (NFJS, OMIM tion worsens during warm humid climate, with most common 161000) is a rare autosomal dominant disorder, characterized by complications presenting as secondary bacterial infections of palmo-plantar keratoderma, decreased sweating and a reticulate blister lesions on the extremities. Some affected individuals suf- pattern of hyperpigmentation of the skin that occurs with age. fer from focal keratoderma. Mutations in EBS-loc are most fre- Clinically, the disease is mostly manifested by the absence of der- quently distributed in four clusters lying outside of the helix matoglyphs, reticular or mottled hyperpigmentation, hypohidrosis boundaries of K5 or K14, including the non-helical L12 linker due to diminished sweat gland function, heat discomfort, hair de- motif, or in the amino-terminal homologous domain (H1) of fects, nail dystrophy, enamel (teeth) defects. Also, moderate pal- K5, or in the 2B segment of K14 (see www.interfil.org; [8]. How- mo-plantar hyperkeratosis may co-exist with punctate keratoses ever, exceptions do exist and patients with EBS-loc have been showing a linearized pattern [44]. NFJS is usually caused by hetero- identified with mutations in the conserved 1A and 2B helix hot- zygous nonsense or frameshift mutations in the KRT14. The disease spots [31,32,39]. Ultrastructural abnormalities of the cytoskele- is allelic to dermatopathia pigmentosa reticularis (DPR) whose dis- 128 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 tinguishing features include the life-long persistence of skin hyper- mutations have been associated to the non-helical head (E1/V1) pigmentation, sometimes accompanied by partial alopecia and ab- domain of the KRT14. Mostly, these mutations result in non- sent dental anomalies [45]. The brown or gray-brown sense-mediated mRNA decay (NMD) or in the synthesis of a short pigmentation localized on the trunk, proximal extremities, axillae, unstable peptide, that results in K14 haploinsufficiency, and has groins and flexures as well as in the periocular and perioral regions been associated with increased susceptibility to tumor necrosis often begins at age 2–10 years. These starts to fade from 15 years, factor-alpha-induced apoptosis [47]. Thus, providing evidence of explaining why patients above 70 years often present minimal or an inherited keratinopathy resulting from impaired regulation of absent pigmentation [46]. apoptosis, as well as the link between EBS-MP and DDD, this also In contrast to KRT14 mutations in severe EBS, which usually af- infers a potential for defective basal keratins to result in both blis- fects the central alpha-helical rod domains, NFJS/DPR-associated tering and pigmentary diseases.

Table 1 Inherited cutaneous keratin disorders due to keratin mutations. AD autosomal dominant, AR autosomal recessive, KIF keratin intermediate, PPK palmo-plantar keratoderma, 2mutations are risk factors but not a causative association.

Human cutaneous keratin disorder Mode of Type II Type I Tissue-specific expressions Clinical characteristics inheritance keratin keratin Epidermolysis bullosa simplex AD K5 K14 Basal keratinocytes of the epidermis Localized, generalized and/or herpetiform blistering, PPK Localized EBS (EBS-Loc) ’’ ’’ ’’ Localized blistering in friction areas as in palms and soles Generalized non-Dowling-meara EBS ’’ ’’ ’’ ’’ Generalized, ’’non herpetiform’’ mild blistering (gen-non-DM-EBS) EBS Dowling Meara (EBS-DM) ’’ ’’ ’’ ’’ Generalized ‘‘herpetiform’’ blistering, may involve nail, oral and other mucosa Epidermolysis bullosa simplex (EBS-AR) AR K14 ’’ Generalized blistering, ichthyotic plaques and may involve extra-cutaneous area, absent/reduced KIF in basal keratinocytes EBS with migratory circinate erythema K5 ’’ Generalized blistering, circinate erythroderma, (EBS-CE) brown hyperpigmentation, reduced KIF in basal keratinocytes EBS with mottled pigmentation (EBS-MP) K5 K14 ’’ Generalized blistering, reticulate/mottled brown hyperpigmentation, KIF aggregation and increased melanosomes in basal keratinocytes Dowling-Degos disease (DDD) K5 ’’ Hyperkeratotic papules, reticulate, progressive and disfiguring hyperpigmentation, primarily in the flexural areas Naegeli–Franceschetti–Jadassohn AD null K14 ’’ Both share reticulate hyperpigmentation of the syndrome (NFJS)/dermatopathia skin, thickening of the palms and soles (PPK), signs pigmentosa reticularis (DPR) of ectodermal dysplasia, such as abnormal sweating and absence of dermatoglyphics Epidermolytic ichthyosis (EI) AD K1 K10 Suprabasal keratinocytes of the Erythroderma, blister formation, development of epidermis hyperkeratosis, PPK Linear epidermal nevus (LEN) AD K1 K10 ’’ Epidermal hamartomas showing somatic mosaicism of epidermolytic hyperkeratosis Epidermolytic ichthyosis (EI) AR K10 ’’ Erythroderma, blister formation, hyperkeratosis Ichthyosis hystrix Curth-Macklin (IHCM) AD K1 ’’ Severe and extensive hyperkeratosis, with or without palmo-plantar keratoderma Diffuse non-epidermolytic PPK (DNEPPK) K1 Upper suprabasal keratinocytes of Demarcated diffuse PPK palmoplantar skin Palmo-plantar keratoderma with K1 Upper suprabasal keratinocytes of Diffuse PPK, erythematous margin tonotubules (PPK) palmoplantar skin Ichthyosis bullosa of Siemens (IBS) AD K2 Upper suprabasal keratinocytes of Superficial epidermal blistering and desquamation, cornifying epithelia absence of congenital erythroderma and hyperkeratosis, affects flexural areas Epidermolytic palmo-plantar K9 Suprabasal layers of palmoplantar Diffuse PPK, sharp demarcation with erythematous keratoderma (EPPK) epidermis (palms and soles) borders, ±EHK/suprabasal KIF clumping Pachyonychia congenita (PC6a, PC16) AD K6a K16 Suprabasal epidermis of palm, soles Thickening of toenails and fingernails, plantar and epidermal appendages e.g. keratoderma, plantar pain, palmar keratoderma, fingernails, toenails pilosebaceous cysts, follicular hyperkeratosis, oral leukokeratosis, hyperhidrosis Pachyonychia congenita (PC6b, PC17) AD K6b K17 ’’ Thickening of toenails and fingernails, plantar keratoderma, plantar pain, palmar keratoderma, pilosebaceous cysts, follicular hyperkeratosis, oral leukokeratosis, hyperhidrosis Focal non-epidermolytic PPK (FNEPPK) K16 Suprabasal layers of palmoplantar Focal PPK with oral, genital, and/or follicular lesions epidermis (palms and soles) Monilethrix AR, co- K81, Hair shaft (clumps of the structural Hair fragility and total or patchy alopecia, hair shaft dominance 86, 83 proteins) deformation Pseudofolliculitis barbae (PFB) K752 Hair follicle root sheath Hair follicles ingrowth, follicular infections, affects mainly black individuals, inflammatory papules and pustules Ectodermal dysplasia of hair and nail type AD/AR K85 Hair shaft Hypotrichosis and nail dystrophy, associated with keratoderma and ichthyosis, hair scalp fragility J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 129

Dowling-Degos disease (DDD) – disease of K5 ciated with rapid healing of denuded areas with recurrent episodes Dowling-Degos disease (DDD) is a rare genodermatosis inher- of blisters on a background of erythroderma that may persist ited in an autosomal dominant manner with varying degree of pen- throughout life. Later on in adulthood, blistering becomes infre- etrance and is caused by mutation in the KRT5 [48,49]. The disease quent; hyperkeratotic plaques with verrucous scales, mainly is manifested during adulthood often in the 40s, with clinical fea- involving flexural and intertriginous areas develop, but can also tures including small rounded reticulate freckle looking hyperpig- appear on the scalp, neck and infragluteal folds. In most of the pa- mented macules often in the flexural areas including the armpits, tients, palmo-plantar hyperkeratosis is present and bacterial colo- neck, under mammary folds and in the groins or the gluteal folds. nization of the macerated scales causes a distinct foul odor [57– Other involved areas may include the scalp, trunk and arms [49]. 61]. As mentioned, the cutaneous pathology in EI results from Major histopathological characteristics include acanthosis and the expression of abnormal K1 or K10 proteins [62], but contrary papillomatosis, with a digitiform (fingerlike) pattern of dermal to the deep blisters found in EBS, blistering is more superficial EI. papillae, epidermal keratin cysts and dilatation of keratinized hair In EI disease, there is increased proliferation of suprabasal kerati- follicles. It present with diffuse deposits of melanin in the basal nocytes that results in the formation of ichthyosiform lesions as epidermal layer in addition to diverse amount of dermal papillary opposed to herpetiform lesions seen in EBS-DM. Ultrastructurally, melanophages and superficial perivascular infiltration of lympho- basal keratinocytes appear normal, but irregularly shaped patho- cytes. In some cases genital involvement has been reported often gnomonic KIFs aggregates are identified in suprabasal keratino- with progressive and symmetric pigmentation [49,50]. The incon- cytes, appearing as dense peri-nuclear shells as the primary sistency in arriving at a consensus in classifying hereditary pig- event, with secondary suprabasal cytolysis, blistering and hyper- mentary dermatoses, has made Dowling-Degos disease to be keratosis [42]. defined as a clinicopathological entity with several subtypes Similar to EBS, the majority of disease-causing mutations in EI including the Galli–Galli disease, an acantholytic form and the are missense mutations that usually occur within the highly con- reticulate acropigmentation of Kitamura, the acral form of the der- served sequence of the alpha-helical rod and the non-helical H1 matosis [48,49,51]. Molecular genetic analysis has identified dele- domains of K1 and K10. Also, the milder variants of the disease tion, duplication, and nonsense mutations resulting in premature have been associated with mutations located outside the helix termination codon (PTC) in DDD with K5 null mutations [52,53]. boundary motifs or in the L12 linker region [63]. Analogous to However, the dominant inheritance pattern in DDD is often due EBS disorders, the positions of the mutations along the keratin to K5 haploinsufficiency instead of the dominant-negative K5 polypeptides, the degree of disruption and the level of expression mutations. In the acantholytic variant of DDD, the Galli–Galli dis- of the mutated KRT1 and KRT10 genes usually explain the clinical ease [48], Sprecher et al. identified a disruptive KRT5 initiation co- features of EI as well [64–66]. don mutation that results in either the synthesis of a K5N-deletion In EI, rare dinucleotide alterations in KRT10 [64,67] including mutant or haploinsufficiency [54] (see Table 1., and reviews spontaneous de novo point, deletion, deletion vs insertion and [12,23,27] for other forms of EBS). Conclusively, identification of splice site mutations in KRT1 and KRT10 have been associated with novel mutations, genotype–phenotype correlations, disease modi- half of the reported cases [66,68]. It is well established that the nat- fiers and in-depth molecular pathogenesis in EBS will allow im- ure of the mutations can predict the disease phenotype and is proved understanding of the disease pathogenesis and futuristic known that while KRT1 mutations are associated with palmo-plan- development of treatment as well as better patient management. tar keratoderma, KRT10 mutations are associated with the non-pal- moplantar variants [56]. Such assertion of association appears to be unique for KRT1 mutations; but exceptions do exist where Keratinopathic ichthyosis (KPI) – diseases of K1/K2/K10 KRT10 mutations have been identified in patients with severe EI mutations and palmo-plantar keratoderma [68,69]. As in severe EBS, a genetic ‘‘hot spot’’ that affects a highly conserved arginine residue Keratinopathic ichthyosis (KPI), represent a family of ichthyoses (p.Arg156) exist in EI. Also, similar to milder forms of EBS, a mis- occurring due to mutations in keratins referred to as superficial sense HTP mutation p.Ile479Thr in K1 in some cases have been keratin keratodermas which include epidermolytic ichthyosis (EI; associated with mild ichthyotic phenotype [65] as well as only epi- K1/K10 mutations) and superficial epidermolyic ichthyosis (SEI; dermolytic palmo-plantar keratoderma in others [70]. K2 mutations (ichthyosis bullosa of Siemens) [55]. Based on genotype–phenotype correlation studies, a more com- plex link is known to exist where the genetic background can mod- Epidermolytic ichthyosis (EI) – disorders of K1 and K10 mutations ulate the disease phenotype. A proof of principle is the report illustrating that the conserved 156 codon mutations in KRT10, usu- Epidermolytic ichthyosis (EI; OMIM 113800) is a form of con- ally associated with severe phenotype as well lead to milder EI genital ichthyosis with a prevalence of 1 in 200,000–300,000 peo- phenotype [71]. This is interestingly similar to EBS disorder where ple [56]. EI, also previously known as bullous congenital different nucleotide changes at the same site on the polypeptide ichthyosiform erythroderma (BCIE) or epidermolytic hyperkerato- results in different disease phenotypes, examples are the following sis (EHK), is a relatively rare congenital skin fragility disease which KRT5 mutations K5_p.Iso183 [32,72] and the K5_p.Val186 muta- is predominantly autosomal dominantly inherited [55].EIis tions [73,74]. caused by mutations in either of the genes coding for epidermal Even though EI is usually an autosomal dominant trait, several suprabasal keratins, K1 and K10 expressed in keratinocytes of the recent reports have delineated recessive inheritance pattern, suprabasal layers of the epidermis. involving either donor splice site, nonsense or termination codon These disorders are characterized at birth with generalized mutations [75–77]. Characteristic ultrastructural features include, erythroderma (redness of the skin) (Fig. 3c), with severe blistering sparse, homogenous and amorphous KIFs network associated with accompanying widespread hyperkeratosis or thickening of the keratin aggregates, and nonsense KRT10 mutation leading to a loss uppermost layer of the skin, erosions and peeling of the skin upon of K10 expression [78]. In other cases, homozygous nonsense mild trauma (Fig. 3d). Hallmarks include superficial ulcerations KRT10 mutations have been reported. The heterozygous pheno- that develop on the flexural regions of the skin. As a result of epi- types were non-phenotypic carriers, demonstrating that a normal thelial barrier disruption, neonates with EI are at risk of developing K10 allele is sufficient to maintain a normal KIF network in the ab- electrolyte imbalances, severe infection and sepsis. EI is often asso- sence of mechanical stress [77]. 130 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137

The nevoid variant of EI, also termed epidermal nevus of the mains with the possibility of a Methyl-CpG deamination mutation epidermolytic hyperkeratotic type exists and is characterized by hot spot, K2_p.Glu487Lys. It is often clinically very difficult to ichthyosiform lesions that are usually distributed along the Blas- clearly distinguish mild EI from severe IBS, inferring that molecular chko lines and that alternate with normal skin. The nevoid distri- genetic analysis is a definitive predictive diagnosis. However, IBS bution of EI is due to the mosaicism of K1/K10 mutations. patients presenting with severe skin phenotypes previously clinic- Heterozygous missense mutations in KRT10 were discovered in o-pathologically misdiagnosed as EI were upon molecular genetic skin lesions, which were absent in normal skin and led to the pos- analysis correctly diagnosed [89,86]. In the other hand, a heterozy- tulation that during embryogenesis mosaicism occurred as a result gous missense KRT1 mutation, p.Glu478Asp was reported in a fam- of postzygotic spontaneous mutations in KRT1 and KRT10 [79,80]. ily with mild EI presenting with clinical and histological features Children with full-blown EI, born by EI patients with the nevoid similar to IBS [90]. variant, usually have underlying gonadal and cutaneous mosaicism [12]. Analogously, mosaicism has been reported in EBS and in pal- Palmo-plantar keratoderma (PPK) – disorders of palmoplantar K9 moplantar verrucous nevus [12]. mutations The annular variant of EI also termed cyclic ichthyosis with epi- dermolytic hyperkeratosis (EHK) is also a rare disease that has Palmo-plantar keratodermas (PPK), a group of heterogeneous been reported in only seven families so far. At birth, the individuals keratinopathic genodermatoses characterized by hyperkeratotic show classical EI erythroderma, superficial erosions but with skin confined to the palms and soles, usually clinically grouped improvement of symptoms in early infancy. Characteristic clinical into three distinct patterns: diffuse, focalize and punctate. Based features include flares of polycyclic psoriasiform plaques that per- on the identification of the underlying genetic defects in these dis- sist for several weeks to several months with only benign localized orders, a revised classification now exist as a result of the previous disease in adulthood. Except for palmo-plantar hyperkeratosis, the difficulties in classifying based on phenotypic and morphological skin between the flares usually looks normal. Definitely, the iden- criteria. tification of KRT1 and KRT10 mutations suggested a subtype of EI Epidermolytic PPK (EPPK, OMIM 144200), an autosomal domi- [65,67,81,82]. nant genodermatosis that develops within the first months after birth, typically manifests as diffuse hyperkeratosis strictly confined Ichthyosis hystrix of Curth-Macklin (IHCM) – disorder due to to the palms and soles presenting sharp demarcations with ery- mutations in the V2 domain of K1 thematous borders. Ultrastructurally, studies revealed aggregates of keratins in the palmoplantar epidermis, with varying degree of Ichthyosis hystrix (IHCM) is a rare distinct type of autosomal hyperkeratosis between families and within individuals in a fam- dominant skin disorder characterized by extensive hyperkeratosis, ily. Other hallmarks include flexural finger joints knuckle pad-like with or without palmo-plantar keratoderma, which results from keratoses and nails clubbing [91]. Another disease form, the Unna- heterozygous frameshift mutation in KRT1 [83]. Except for the ab- Thost form, which is often clinically identical to Vörner disease, can sence of epidermolysis, histological features are similar to those of be distinguished histologically and ultrastructurally by the absence epidermolytic hyperkeratosis or other cornification disorders like of EHK and suprabasal KIF clumping, respectively [65]. A spectrum erythrokeratodermia variabilis. Ultrastructural features include of pathogenic substitutions including missense or small in-frame perinuclear vacuolization, binucleated cells and suprabasal non- insertion vs deletion mutations of KRT 9, have been reported in aggregated shell-like tonofilaments. Here, the KRT1 mutations several families with EPPK, with the beginning of the 1A rod do- mostly affect the V2 domain and dramatically alter it biochemical main and the end of the 2B rod domain of the KRT 9 as mutation properties, but does not interfere with KIF formation. Instead, the hotspots [92–96]. In addition, mutations have been identified in mutation is postulated to relate to intracellular mis-orientation the variable V1 and V2 domains, which are responsible for the of loricrin, responsible for the organization of the cornified cell greatest sequence variation in different keratins, and some have envelope [83,84]. Beside, a heterozygous frameshift mutation been associated with non-epidermolytic palmo-plantar kerato- was identified in the V2 domain in close proximity to the IHCM derma [87]. Another rare PPK ultrastructurally characterized by mutation in KRT1, which results in striate palmo-plantar kerato- of the presence of ‘‘tonotubules’’, first described in a large German derma (SPPK), a heterogenetic disorder associated with pathogenic family, called PPK with ‘‘tonotubular’’ keratin, has been described mutations in KRT1, which is occasionally associated with muta- in five families [97]. Molecular analysis revealed two missense tions in desmosomal proteins, desmoglein 1 and desmoplakin. mutations closed to the 1B domain of K1 [98,99]). As these tubular Consequentially, this frameshift mutation lead to partial loss of structures have not been described in other keratin disorders, they the glycine loop motif in the V2 domain of KRT1 [85], and therefore suggest a different role for the 1B domain in the formation of KIF. suggests a distinct pathogenic pathways in the tail domain of KRT1.

Ichthyosis bullosa of Siemens (IBS) – disorders linked to K2 mutations Keratin defects in skin appendages (hair and nail) and other genodermatoses Ichthyosis bullosa of Siemens (IBS, OMIM 146800), a more superficial epidermolytic ichthyosis (SEI), is an autosomal domi- Monilethrix – disease of the hair K81/K83/K86 mutations nant keratinization disorder, with similar but milder clinical fea- tures compared to EI. Clinical characterized include, the absence Monilethrix is a rare predominantly autosomal dominant disor- of congenital erythroderma and hyperkeratosis and is mostly local- der that affects the hair with characteristic shaft anomalies, featur- ized to flexural areas. The blistering is more superficial and has ing uniform elliptical nodes with intermittent constrictions been called molting (Mauserung), and the granular degeneration resulting in hair fragility and diffuse or patchy alopecia. Inheri- is restricted to the uppermost spinous and granular layers with tance is usually accompanied by a high penetrance and variable characteristic desquamation that result in denuded skin areas expressivity with a few reports of autosomal recessive and spo- [86–88]. Aggregates of keratin filament bundles and superficial radic cases [100]. Clinical features include a small area of hair dys- cytolysis often correspond to the epidermal tissue layer of K2 trophy to total alopecia, hair shaft deformation with characteristic expression. The majority of pathogenic mutations affect the HTM moniliform appearance caused by the nodes and internodal thin- of K2 although mutations have been reported in the 1A and 2B do- ning of the shaft, and abnormalities of cuticle, cortex and keratiniz- J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 131

Fig. 4. Clinical features of pachyonychia congenita (PC) affecting the plantar epidermis and the nails with varying severity and keratin gene mutations. (a) Patient (PC-17) presenting with mild plantar keratoderma due to K17 (K17_p.92Asn>Ser) mutation. (b) Patient (PC-16) with severe and painful plantar keratoderma due to a K16 (K16_p.124Leu>Pro) mutation. (c) Patient (PC-16) with mild to severe hypertrophic nail dystrophy with a K16 (K16_p.132Leu>Pro) mutation. (d) Patient (PC-6a) with severe hypertrophic nail dystrophy involving the nail beds and having a K6a (K6a_p.171 Asn>Lys) mutation.

ing zones of hair follicles. The internodes are characterized by nance [107,108]. Multiple cases with autosomal recessive inheri- wrinkled cortical cells leading to hair fragility, and lack of medulla tance and mutations in desmoglein 4 (DSG4), a cadherin [101]. Invagination of the cuticle cells into the cortex, degenera- subfamily member expressed in the hair cortex and upper cuticle, tion, cytoplasmic vacuolation and abnormal tonofibrils are seen have been reported [109–111]. Mutated DSG4 is now associated in cortical cells which are particularly affected in the hair matrix with localized autosomal recessive hypotrichosis, and with the [102]. In addition to the clinical signs of short, sparse, fragile, non- great variability in the moniliform trait, this suggests a clinical growing hair, patients may present with keratosis pilaris, koilo- overlap between monilethrix and localized autosomal recessive nychia with rare, systemic problems such as mental and physical hypotrichosis. retardation, syndactyly, cataract, teeth and nail dystrophies [103,104]. Improvements during adolescence and pregnancy have Pseudofolliculitis barbae – disease due to predisposition to K75 been documented, suggesting a hormonal influence. Light micros- polymorphism copy is usually diagnostic with the hair showing a typical monili- form (beaded) structure. Ultrastructural findings show cortical Pseudofolliculitis barbae (PFB), is a common hair disorder with defects and aggregates of hair shaft structural proteins [7105] sug- chronic, irritating, and potentially disfiguring condition that usu- gesting trichocyte keratins as the prime disease candidates. Patho- ally develops after shaving hair from predilection sites (beard genic mutations have been delineated in genes that code for the area), leading to hair follicles ingrowth and follicular infections basic hair keratins K81, K83 and K86 in humans, often presenting [112]. Incidence rate of the disorder is difficult, but some studies normal lanugo hair in neonates [104]. Mutations in the HTM of showed that it can affect up to 1 out of every 5 caucasian individ- KRT86 (previously denoted KRTHB6), in K86 (Hb6; 90%) and in uals and often occurs much more commonly in black persons due HTM of KRT81 (Hb1; 100%) in K81 have been identified [106]. Ker- to a genetic predisposition to curled hair. Clinical features include atin K83 (Hb3) is co-expressed with K81 (Hb1) and K86 (Hb6) in the appearance of inflammatory papules and pustules. Molecular the mid-cortex and shares an almost identical rod domain se- analysis in severely affected males and asymptomatic females re- quence, but only one mutation in the 2B region (p.Glu407Lys) vealed a single-nucleotide polymorphism that results in a disrup- has been identified to date in a monilethrix family with co-domi- tive p.Ala161Thr substitution in the 1A alpha-helical segment of 132 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137

K75. This has been suggested to be partially responsible for the Table 2 phenotypic expression and represents an additional genetic risk Summary of the percentage contribution of the different ectodermal tissues in PC disease severity, including the percentage of the different PC-related keratin genes factor for PFB [113]. K6a, K6b, K16 and K17 mostly expressed in suprabasal layers of palmoplantar skin, as well as epidermal appendages and oral mucosa. Additional unpublished data from International PC Research Registry (IPCRR) shows that in 487 genetically tested patients more than 90% of the disease-causing PC mutations are associated with the Pachyonychia congenita (PC) – disorder of plantar and nail K6/K16/ four ‘‘classic’’ PC keratins namely: about 49% are K6a, 8% are K6b, 27% are K16 and K17 mutations 16% are K17 while 10% of patients diagnosed with PC have other non-PC mutations (such as Connexin, PPK, etc.) or mutations yet to be discovered.

Pachyonychia congenita (PC; OMIM 167200 and 167210), is a IPCRR data November Pachyonychia congenita (PC) Total rare autosomal dominant genodermatosis chiefly affecting tissues 2010, N = 271 Percentage of the different keratin N = 271 of the ectodermal origin such as the palmoplantar epidermis, nail Genetically analyzed genes involved (%) bed, mucosae and the pilosebaceous unit. The fundamental clinical K6a K16 K6b K17 features are painful palmoplantar (preponderantly plantarkerato- N = 125 N =79 N =20 N =47 derma) (Fig. 4a and b); hypertrophic nail dystrophy (Fig. 4c and Thickening of toenails 100 94 100 100 98 d); oral leukokeratosis; and a variety of cysts arising from hyper- Thickening of fingernails 100 73 50 91 87 Plantar keratoderma 89 100 100 81 92 keratosis of pilosebaceous apparatus. Occasionally, plantar epider- Plantar pain 98 95 100 89 96 mal blistering with varying degree of involvement of the skin Palmar keratoderma 54 84 30 47 59 (including cysts and follicular hyperkeratosis), larynx, oral mucosa, Cysts 78 24 55 96 63 teeth and tongue [114,115]; Wilson2; Eliason3. PC is often inherited Follicular hyperkeratosis 74 05 35 81 52 Natal/prenatal teeth 03 00 0.0 81 15 in an autosomal dominant manner and is caused by pathogenic Oral leukokeratosis 94 58 35 30 68 mutations in at least four keratin (KRT) genes (KRT6A, KRT6B, Ear – short/sharp pain 21 08 10 02 13 KRT16, and KRT17) that encode the keratins K6a, K6b, K16, and Ear – excessive wax 41 20 35 28 32 K17, respectively, [115]. Previously, based on minor phenotypic dif- Larynx – hoarsness 43 09 25 26 29 ferences, classification distinguished two PC subtypes (PC-1, or Hyperhydrosis 50 43 50 49 48 Jadassohn-Lewandowski subtype; and PC-2, or the Jackson-Lawler subtype), chiefly based on the presence or absence of pilosebaceous cysts [114,115]. This classification was based on clinical, but non- the individual patient lifestyle and care they take of their lesions genetically analyzed cases, but with the advent of data from studies and keratin fragility from specific mutations (Fig. 5). analyzing hundreds of patients in the International PC Research Reg- The IPCRR, has to date registered over 478 families including istry (IPCRR), presenting intriguing phenotypic overlap between PC about 1000 individuals, with 199 genetically tested families. Previ- subtypes, the limitations of this previous classification has became ous and recent findings have reported several molecular genetic re- clear. For instance regardless of the genotype, many PC patients have sults [115–123], and a recent data on 90 new families will soon be some form of epidermal cysts (Wilson1). Based on the IPCRR data, a published (Wilson2). novel consensus classification based on the mutated gene and their Together, these findings have identified PC associated mutation clinical subtype has been adopted [116]. This new consensus classi- hotspot codons which may be useful for future development of tar- fication categorizes PC as PC-6a, PC-6b, PC-16 and PC-17, where for geted therapies. There is a wide a spectrum of keratin mutations in instance PC-6a indicates a patient with a mutation in the gene PC. Splice site dominant-negative mutations have been reported encoding, K6a etc. For complete dataset on this novel consensus which may likely lead to nonsense-mediated mRNA decay, and loss nomenclature see Wilson1 and Eliason2. Even though the clinical of expression of mutated allele. Similar to certain cases of KRT5 and feature that resulted in naming of the condition is hypertrophic nail KRT1, mutations in genes closely related to KRT6A have been re- dystrophy, focal plantar keratoderma associated with severe debili- ported to affect the intron 1 splice sites [124,125]. Analogous to tating pain is the major problem encountered by PC patients, with K5 mutation in EBS [126,127], heterozygous nonsense mutations serious negative impact on quality of life (Eliason2). Similar to other have been identified in PC predictive to lead to expression of a keratin genodermatoses discussed above, most pathogenic PC-re- truncated dominant-negative K6a protein, lacking the end of the lated keratin mutations are heterozygous missense mutations or tail domain, expected to escape NMD and express mutant polypep- small insertion/deletion mutations mostly inherited as an autosomal tide [128]. Analogously, premature termination codon mutations dominant trait, for literature see www.interfil.org and [8]. These have been reported in other dominant keratinopathies, e.g. K5 in mutations renders cytoskeletal fragility causing cytolysis and hyper- Dowling-Degos disease as discussed [52,53] and in K14 in NFJS keratosis of the basal/suprabasal layers of palmoplantar skin, as well [45] both as already discussed above. as epidermal appendages and oral mucosa, predominantly express- ing K6a, K6b, K16 and K17 [28]. The severity of the clinical features Ectodermal dysplasia of the hair and nail type keratins of PC can vary quite widely both among and within families and based on recently updated information from 271 genetically ana- Ectodermal dysplasia (ED) is a congenital syndrome character- lyzed PC patient samples in the International PC Research Registry ized by abnormal development of tissues of ectodermal origin such (IPCRR) showing differential involvement of the various PC associ- as skin and its appendages (the hair, nails, teeth and sweat glands). ated keratin and their respective tissue involved (Table 2, IPCRR data There exist about 200 known ectodermal dysplasias with about 30 on file November 2010). genetically tested and characterized at the molecular level. The The reason for this variability is not known, but is likely a com- clinical characteristics and the molecular basis of ED are poorly bination of genetic/epigenetic and environmental factors including understood. Most of the hair and nail type ectodermal dysplasia (HNED; OMIM 602032), the congenital disorder characterized by 2 N.J. Wilson, S.A. Leachman, C.D. Hansen, A. McMullan, L.M. Milstone, M.E. hypotrichosis and nail dystrophy, can show either an autosomal Schwartz, W.H.I. McLean, P.R. Hull, F.J.D. Smith, A large mutational studyin pachy- dominant [129] or autosomal recessive [130,131] pattern of inher- onychia congenita (submitted for publication). itance. They are mostly associated with abnormalities, such as ker- 3 M.J. Eliason, S.A. Leachman, B. Feng, M.E. Schwartz, C.D. Hansen, A review of the clinical phenotype in 254 patients with genetically-confirmed pachyonychia congen- atoderma or ichthyosis, skeletal anomalies, cardiac irregularities, ita (submitted for publication). mental or psychomotor retardation and some hair scalp fragility. J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 133

Fig. 5. Triton extracted monolayer cultures of keratinocytes cytoskeleton and 3-D epidermis engineered with keratinocytes harboring mutations. Heat stress of keratin cytoskeleton reveals pathognomonic keratin-aggregates in keratinocytes harboring (a) K5_p.475Gly>Glu mutation in severe generalized EBS-DM keratinocyte cell line (EB11) when stained with keratin 5 antibody, (b) K10_p.156Arg>Gly mutation in a moderate EI-phenotype keratinocyte cell line (EH21) when stained with keratin 10 antibody. Keratin 10 expression was induced in submerged culture by calcium induced differentiation of keratinocytes. (c) Hematoxylin staining of organotypic epidermis generated using immortalized epidermolytic ichthyosis (EI:EH31) keratinocyte cell lines derived from a severely affected patient’s organotypic epidermis and without heat-stress. The reconstructed epidermis here shows well-defined basal layer, superficial cleft or cytolysis at the suprabasal layers (arrows) leaving unperturbed basal and cornified layers, a histological feature that mimics the in vivo phenotype of the patient. (d) Hematoxylin staining of organotypic epidermis generated using immortalized epidermolysis bullosa simplex (EB11) keratinocyte cell lines derived from a severely affected EBS-DM patient’s organotypic epidermis and after heat-stress. The organotypic epidermis shows well- defined basal layer with some basal cell cytolysis or cleft formation reminiscent of in vivo severe generalized EBS phenotype. All organotypic epidermis was harvested after 12 day post-lifted at air–liquid interface.

Ultrastructural examination shows inconsistent thickness of the and bio-engineering models of the affected skin. A significant in- hair shaft, without periodic variation in hair diameter as observed crease in the knowledge that governs the biology and pathophysi- in monilethrix hair [102]. A homozygous mutation in the type II ology of keratinopathic genodermatoses has benefited hair keratin gene KRT85, has been identified in a consanguineous tremendously from the development and use of knockout and Pakistani family with autosomal recessive PHNED [132]) [132].In transgenic mice models [136–138], and more recently in vitro two other consanguineous Pakistani families with PHNED two dis- models of such disorders. tinct homozygous mutations in the KRT85 gene were disclosed in Animal models of keratin diseases have been useful for under- both families with an autosomal recessive inheritance [133]. How- standing the function of keratins, to identify and delineate molec- ever, only five families have been reported with two distinct KRT85 ular defects in human keratinopathic-disorders, and to develop mutations so far, making genotype–phenotype correlations for potential therapies. Several native pathogenic mutations with blis- KRT85 mutations more difficult to ascertain. In a cohort, some pa- tering phenotypes have been identified in other animals, such as tients exhibited severe hair and nail phenotypes, suggesting a more dogs, horses and sheep [139], most of which have provided in- disruptive potential for the p.Arg78His mutation compared to the depth understanding of the role of animal models in disease path- mutation p.Pro483ArgfsX18 mutation in the K85 protein. The ogenesis. Three main groups of animals exist: animals in which a K85 is expressed in the lowermost matrix, precortex, cortex, and specific keratin genes have been deleted [140–142]; animals cuticle of the hair shaft (follicle) [134], as well as in the basal com- expressing keratins into which mutations have been introduced partment and in the lower keratogenous zone of the apical and [138,143]); animals in which the expression pattern of keratin ventral nail matrix [135]. It has been suggested that genetic dys- has been altered either by over-expression of normal keratin function in K85 can disrupt KIF formation resulting in abnormal [144] or by the use of promoters to direct keratin expression to a desmosomal assembly in hair and nails [133]. Therefore, the ab- different tissue or group of cells [145]. Most human keratin disor- sence of the protein during hair and nail formation can explain ders are dominant disorders resulting from missense mutations, the severe hair and nail phenotype [7]. whereas most transgenic mice are keratin knockouts with reces- sive phenotypes and thus their phenotype may not be directly comparable to those of humans. To date, a plethora of evidence Animal and in vitro models of keratin-related cutaneous providing intriguingly new insight in the complexity of the effects diseases mediated by signaling pathways, which result in phenotype mod- ification in diseases are still emerging. A prototype example is the The molecular characterization and assessment of therapeutic cell-mediated inflammation in keratin 5-deficient mice [36]. outcomes for inherited cutaneous disorders requires faithful pre- Genetically engineered mouse models have been generated for clinical models such as animal models and in vitro proliferating the major EBS, EI and PC and reviewed in [146]. 134 J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137

In vitro models of inherited keratin disorders provide useful sys- ing the effects of new drugs on animal models and clinical develop- tems with which to elucidate the cellular, morphological, biochem- ment. These cells were able to differentiate and reconstitute a 3-D ical and biophysical mechanisms in the disease pathogenesis, and engineered epidermal tissue in vitro on cell culture inserts or de- represent an initial step towards the development of novel thera- epidermalized dermis. It was shown that cells from patients with peutic strategies. Since introducing mutant keratins into keratino- severe disease recapitulated the histological and phenotypic alter- cytes lead to positive keratin-aggregates formation, often with ations reminiscent of the in vivo donor phenotype [152,159], negligible correlation between in vitro and donor phenotype sever- (Fig. 5c and d), a phenomenon consistent with a previous report ity, alternative strategies created finite and permanent cell lines in an in vitro recessive dog model of EI [152,160,161]. Recently, from donor patients with known pathogenic keratin mutations. organotypic epidermal model was successfully used to evaluate Primary patient-derived keratin-defective keratinocytes have been the effectiveness of ‘‘self-delivery’’ siRNA. The end point was the used as model for studying the impact of heat-stress induced ker- reduction of pre-existing fluorescent reporter gene expression atin aggregation [72]. compared to the minor effect in controls conditions. Also, a marked However, primary keratinocytes exhibited finite replicative life- reduction of mutant keratin mRNA expression compared to the spans in culture and developed heterogeneous behavior with wild-type was observed [162]. Additionally, a recent approach ap- increasing passage numbers, a limitation that makes them subop- plied a combination of in vitro bio-engineered skin and engraft- timal for long-term reproducible and more extensive functional ment onto immuno-compromized mice (the skin-humanized evaluations, especially when testing new treatment rationales. mouse model) [163–165], which has been developed and is ame- Complementing such knowledge with biochemical, biophysical nable for genetic intervention to clinically treat PC [166]. Thus, and pharmacological assays using patient-derived cells culture these models represent useful tools for future development of clin- models will rapidly enhance development of therapy. ical remedies for keratin-related skin disorders. Established immortalized keratin-mutant keratinocyte cell lines are invaluable tools required at early stage for devising therapeutic Application of animal and in vitro models strategies, and the phenotypic rescue of inherited skin disorders using genetically manipulated cultured cells have shown proofs The development of new bio-engineered skin systems in combi- of principle [147–149]. To date, several immortalized patient-de- nation with establishing a robust humanized skin approach, pro- rived keratin defective cell lines have been established as long- vide an optimized translation of human skin physiology and term reproducible cellular models of EBS, EI and PC [150–154]. Fu- pathophysiology. Thus, the blend of regenerating human skin in ture challenges remain to rapidly make use of such established immunodeficient mice following bio-engineered skin (in vitro) models to further explore the detailed pathomechanisms, test grafting appears a perfect correlation between animal, in vitro new treatment options and develop tools with which to curb cur- and in vivo fidelity in translational medicine [163,164]. Such com- rent treatment limitations for these disorders. bination of organotypic epidermis and skin-engrafted mouse mod- els may generate large numbers of keratin-defective skin- Effects of keratin mutations and physical stress on cytoskeleton engrafted mice [163–165], potentially enabling the test of novel resilience in keratin-defective cells gene-based or pharmacological therapy for these untreatable skin-disorders, and recent report already showed proof of principle Analysis of primary and immortalized keratin-mutant keratino- [162,167]. cytes demonstrated some common features as well as certain divergence related to the phenotypic variation seen in the patients in vivo. These changes could presumably be due to an increased Problems related to treatment of keratin genodermatoses and physiological stress burden on the severely mutant cells, incurred prospects for emerging therapies by the requirements of handling mutated keratins [72,153,155]. It has also been observed that when keratin-defective cells are Currently no curative therapy for keratin-related cutaneous dis- grown in conditions favoring desmosomal connections (serum orders exists, and some may argue that current therapies are te- containing medium) as well as at increased confluency; they are dious, only moderately effective and involve significant risks of more resistant to the effect of physical stress. This emphasizes side-effects. However, therapy development for some keratinopa- the necessity of uniformity in cellular confluency when performing thies has improved considerably over the years and this is becom- such experiments. Cells grown in culture have been extremely use- ing obvious for EBS, KPI and more recently, PC. Unfortunately, it ful for the establishment of different functional assays such as heat, was thought that most of the hurdles lie in the lack of in-depth mechanical and osmotic shock, which explore the effects of stress- understanding of the detailed molecular signature of the defect. induced KIF remodeling. Basal/suprabasal keratinocyte expressing This is not the case now as significant knowledge is available, yet keratin mutations revealed pathologic keratin positive aggregates most of the treatment outcomes are still below expectation. There in both EBS and EI disease models in vitro (Fig. 5a and b) is an obvious need for more research on the disease mechanisms [151,154–158]. Moreover, the effect of stress on mutant keratin and new therapies for these discomforting genodermatoses, aim- aggregation in both basal and suprabasal keratin defective cell ing for best restoration of near normal to normal functioning of lines were shown to be similar to those of their respective primary the skin and its appendages. The severe forms of keratinization dis- cells [152]. These data infer that immortalization did not seem to orders represent a serious handicap, requiring life-long, manage- interfere with the intrinsic properties of the patient-derived pri- ment, therapy and affecting the patient’s quality of life. mary keratinocytes, and thus point out the importance of the data Associated complications, such as heat intolerance owing to provided by experiments on these cell lines. chronic skin infections mainly in EI patients for instance, add to the incapacitation of the patients and often require constant med- In vitro tissue engineering of keratin defective epidermis ical attention. Therefore, medical and ethical considerations, con- comitant with funding agencies should support the development Since keratin diseases are tissue fragility disorders, which may of experimental approaches to treatments. These may include not be adequately reproduced in monolayer cultures, attempt to cutaneous gene therapy, natural products, small molecule-based mimic the disease phenotype in reconstructed epidermis and other pharmacological and drug-based discovery therapy approaches to dermal equivalent substrates in vitro, has been made prior to test- overcome treatment limitation thus helping this group of patients. J.C. Chamcheu et al. / Archives of Biochemistry and Biophysics 508 (2011) 123–137 135

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