Palmoplantar keratodermas are a heterogeneous group of disorders characterized by abnormal thickening of the volar epidermis (Blaydon and Kelsell, 2014Blaydon D.C. Kelsell D.P. Defective channels lead to an impaired skin barrier.J Cell Sci. 2014; 127: 4343-4350Crossref PubMed Scopus (25) Google Scholar, Maruthappu et al., 2014Maruthappu T. Scott C.A. Kelsell D.P. Discovery in genetic skin disease: the impact of high throughput genetic technologies.Genes. 2014; 5: 615-634Crossref PubMed Scopus (9) Google Scholar). A subset of palmoplantar keratodermas are associated with syndromes linked to other cutaneous features (Betz et al., 2012Betz R.C. Cabral R.M. Christiano A.M. Sprecher E. Unveiling the roots of monogenic genodermatoses: genotrichoses as a paradigm.J Invest Dermatol. 2012; 132: 906-914Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and also noncutaneous conditions such as hearing loss, cardiomyopathy, and esophageal cancer (Blaydon Diana et al., 2012Blaydon Diana C. Etheridge Sarah L. Risk Janet M. Hennies H.-C. Gay Laura J. Carroll R. et al.RHBDF2 mutations are associated with tylosis, a familial esophageal cancer syndrome.Am J Hum Genet. 2012; 90: 340-346Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Kelsell et al., 2001Kelsell D.P. Di W.-L. Houseman M.J. Connexin mutations in skin disease and hearing loss.Am J Hum Genet. 2001; 68: 559-568Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Palmoplantar keratodermas specifically associated with defects in hair development include the desmosomal disorders linked to phenotypes such as woolly hair and alopecia (Brooke et al., 2012Brooke M.A. Nitoiu D. Kelsell D.P. Cell-cell connectivity: desmosomes and disease.J Pathol. 2012; 226: 158-171Crossref PubMed Scopus (133) Google Scholar). Two adult siblings from a consanguineous family of Pakistani origin, whose parents were first cousins, presented with an autosomal recessively inherited palmoplantar keratoderma, leukonychia, and exuberant curly scalp hair (Figure 1a). Both affected individuals described the progressive development of yellowish thickened scaly skin affecting the palms and soles since 2 years of age, and toenail dystrophy in their teenage years. Examination revealed marked diffuse, verrucous hyperkeratosis with deep fissuring affecting the soles (Figure 1a) and to a lesser extent, the palms. There was no evidence of transgradiens. The toenails were dystrophic with onycholysis and leukonychia was also present, most evident in the finger nails. Onychomycosis was excluded by negative fungal culture. No abnormalities of teeth or sweating were identified. The siblings also described having extremely thick, rapidly growing curly scalp hair since childhood, but without excessive hair growth elsewhere. Neither parent had a similar hair or skin phenotype, and they had no other offspring. Clinical photographs were obtained, and written consent was provided by patients for their publication. Blood samples were collected after written informed consent in adherence with the Declaration of Helsinki principles and approval of the East London and City Health Authority. Whole-exome capture from both siblings was performed using SeqCap EZ Human Exome Library v2.0 (Roche NimbleGen, Madison, WI) and sequenced with 100-bp paired-end reads on the HiSeq 2000 platform (Illumina, San Diego, CA). Resulting reads were mapped to the hg18 human reference genome using the Novoalign alignment tool (Novocraft Technologies Sdn Bhd, Selangor, Malaysia). Sequence variants were called with SAMtools and annotated with ANNOVAR (Wang et al., 2010Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7948) Google Scholar). Given the history of parental relatedness, the variants were filtered for homozygous changes shared by the two affected individuals, of which 83 homozygous variants reported either as novel or with an Exome Variant Server (EVS) (NHLBI GO Exome Sequencing Project, Seattle, WA) estimated frequency of less than 0.01 were selected as potential candidates. These candidates included a homozygous C to A transversion, c.C101A, in exon 2 of FAM83G (NM_001039999), a gene reported to be mutated in hereditary footpad hyperkeratosis in Kromfohrländer and Irish terrier dog breeds, which presents with fissuring hyperkeratosis of the paws and a bushy coat (Drogemuller et al., 2014Drogemuller M. Jagannathan V. Becker D. Drogemuller C. Schelling C. Plassais J. et al.A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH).PLoS Genet. 2014; 10: e1004370Crossref PubMed Scopus (35) Google Scholar, Sayyab et al., 2016Sayyab S. Viluma A. Bergvall K. Brunberg E. Jagannathan V. Leeb T. et al.Whole-genome sequencing of a canine family Trio reveals a FAM83G variant associated with hereditary footpad hyperkeratosis.G3 (Bethesda, Md). 2016; 6: 521-527Crossref PubMed Scopus (16) Google Scholar). Furthermore, the bushy hair phenotype of the “woolly” mice (wly mouse) has been linked to a 995-bp deletion in fam83g (Radden, 2013Radden IInd, L.A. Child K.M. Adkins E.B. Spacek D.V. Feliciano A.M. King T.R. The wooly mutation (wly) on mouse chromosome 11 is associated with a genetic defect in Fam83g.BMC Res Notes. 2013; 6: 189Crossref PubMed Scopus (11) Google Scholar). Therefore, the variant in FAM83G presented as an obvious candidate for further analysis. The homozygous c.C101A variant in FAM83G was confirmed by Sanger sequencing in both siblings, and both parents were heterozygous carriers (primer sequences: FAM83G-F: 5′ CCGGGCTCATCAGGTCTTT 3′ and FAM83G-2R: 5′ GAGCGGTCCGACTTCTGG 3′; Figure 1b). The c.101C>A mutation results in loss of a Cac8I restriction endonuclease consensus site and segregation of the mutation with the condition was confirmed by restriction fragment length polymorphism analysis (Figure 1c). The mutation c.C101A is predicted to change an evolutionary conserved alanine to glutamate (p.A34E) in the protein FAM83G. This missense mutation was absent from the database of Single Nucleotide Polymorphisms (dbSNP), ExAC, 1000 genomes, gnomAD, and EVS. It is predicted to be deleterious to the protein structure (Polyphen2 score 1.00, SIFT score 0.00). This residue is conserved across vertebrates (Figure 1c) as well as between members of the FAM83 family of proteins. The FAM83A-H family is characterized by the presence of a conserved N-terminal domain of unknown function: DUFI 669. Like the missense variant, p.A34E, reported in this study, the missense variant, p.R52P, underlying hereditary footpad hyperkeratosis in dogs is also located in the DUFI 669 domain. FAM83G expression is specifically enriched (>fivefold) in the skin compared with 26 other tissue types (Edqvist et al., 2015Edqvist P. Fagerberg L. Hallstrom B. Danielsson A. Edlund K. Uhlén M. et al.Expression of human skin-specific genes defined by transcriptomics and antibody-based profiling.J Histochem Cytochem. 2015; 63: 129-141Crossref PubMed Scopus (49) Google Scholar). FAM83G has been implicated as a regulator of BMP signaling (Vogt et al., 2014Vogt J. Dingwell K.S. Herhaus L. Gourlay R. Macartney T. Campbell D. et al.Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling.Open Biol. 2014; 4: 130210Crossref PubMed Scopus (28) Google Scholar). A skin biopsy obtained from one of the siblings revealed acanthosis of the epidermis (Figure 1e), whereas immunohistochemistry demonstrated a marked reduction in FAM83G (Abcam ab121750, Cambridge, UK) expression compared with control skin (Figure 2a). Upregulation of Ki67 (Abcam ab15580) was evident, and keratin 14 expression (Abcam LL001) was not restricted to the basal layer suggesting dysregulated proliferation. As keratinocyte and hair growth development has been linked to Wnt signaling, immunohistochemical staining of β-catenin (BD Transduction Labs 610153, Oxford, UK) was performed and increased levels of active β-catenin were seen within cell nuclei of the patient epidermis compared with control (Merck Millipore 05-655 Clone 8E7, Watford, UK) (Figure 2a and b). As nuclear translocation of β-catenin is induced by activation of Wnt signaling (Greco et al., 2009Greco V. Chen T. Rendl M. Schober M. Pasolli H.A. Stokes N. et al.A two-step mechanism for stem cell activation during hair regeneration.Cell Stem Cell. 2009; 4: 155-169Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar), FAM83G may be a repressor of Wnt signaling and proliferation. To investigate the hair phenotype, hair from one sibling and both parents were obtained. Confocal imaging showed membrane-bound deposits on the outer cuticle of the hair shaft present in the affected sibling but absent in both parents (Figure 2b). Transmission electron microscopy revealed consistent regions of poorly differentiated cuticle cells possibly deriving from the inner root sheath (Figure 2c). FAM83G expression within hair follicles from the back skin of control mice was investigated. FAM83G was found to be expressed during the anagen growth phase (P28) (Figure 2d) and exhibited high levels of staining intensity at the inner root sheath and in the dermal papilla. The expression of FAM83G appeared to be maintained in catagen/early telogen (P42), within the inner root sheath (Figure 2e). FAM83G has previously been identified as a differentially expressed gene in adult mouse hair bulge stem cells compared with dermal papilla cells during telogen (P56) (Greco et al., 2009Greco V. Chen T. Rendl M. Schober M. Pasolli H.A. Stokes N. et al.A two-step mechanism for stem cell activation during hair regeneration.Cell Stem Cell. 2009; 4: 155-169Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Our findings highlight a key role for FAM83G in the homeostasis of the palmoplantar epidermis and hair. Dampened Wnt/β-catenin signaling is a feature of several common forms of hair loss, including alopecia areata and androgenetic alopecia. Further studies of FAM83G, in particular regarding its role in hair biology and the hair cycle, may support targeting FAM83G and associated pathways in scalp hair disorders. David P. Kelsell: http://orcid.org/0000-0002-9910-7144 The authors state no conflict of interest. TM holds a MRC Clinical Research Training fellowship (MR/K002740/1). The study was supported, in part, by the 2016 CHANEL-CERIES research award to DPK.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2 −/− mice compared with irhom2 +/+ mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this ‘stress’ keratin is regulated.

ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1α was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder.

Previously, we have linked point mutations in the rhomboid protein iRHOM2 to tylosis with oesophageal cancer (TOC), a dominantly-inherited disease of palmoplantar keratoderma, oral leukokeratosis and the only known syndrome of inherited oesophageal cancer susceptibility. In keratinocytes, TOC-associated iRHOM2 mutations result in upregulated activity of the broad-spectrum ectodomain sheddase ADAM17 (whose maturation iRHOM2 regulates), and greatly upregulated constitutive shedding of ADAM17 substrates, including EGFR ligands and TNFα. Furthermore, GEO expression analysis shows the iRHOM2-ADAM17 pathway is significantly upregulated in lesional psoriasis and atopic eczema, indicating a role for this pathway in common skin disorders. Using cultures of hyperproliferative TOC-patient-derived keratinocytes, we show by western blot and immunocytochemistry that ADAM17-dependent EGFR ligand hypersecretion drives hyperactivation of EGFR/MAP-kinase signalling. Next, qRT-PCR and ELISA analysis showed that TOC keratinocytes display strong constitutive upregulation of pro-inflammatory cytokines IL-6 and IL-8, and that their expression is sensitive to inhibition of ADAM17 or the EGFR, or knockout of iRHOM2 using CRISPR/Cas9 technology; showing that the iRHOM2-ADAM17-EGFR axis drives pro-inflammatory cytokine secretion. Upregulated pro-inflammatory cytokine production was then shown to dysregulate downstream signalling, with the combination of upregulated secretion of IL-6 (and IL-6R, an ADAM17 substrate) driving strong constitutive upregulation of STAT3 signalling and NFκB activity in TOC keratinocytes. Notably, pharmacological inhibition of either ADAM17 or the EGFR was effective in abrogating the observed upregulation of EGFR/MAP-kinase and STAT3 signalling, and normalising TOC keratinocytes’ hyperproliferative phenotype. Taken together, these findings illustrate a key role for the iRHOM2-ADAM17 axis in growth factor and cytokine regulation in the skin, offering new insights into epidermal signalling networks and mechanisms of hyperproliferation.