GRHL2 mutations were found to cause an autosomal recessive ectodermal dysplasia syndrome in two unrelated Kuwaiti families. Affected individuals presented with nail dystrophy, palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, dysphagia, deafness, and asthma. Whole exome sequencing identified homozygous GRHL2 mutations in both families, demonstrating its role in skin development and human disease. Analysis of patient cells showed altered cell morphology, impaired tight junctions, and cytoplasmic translocation of GRHL2, indicating pathogenic effects of the mutations.
GRHL2 mutations were found to cause an autosomal recessive ectodermal dysplasia syndrome in two unrelated Kuwaiti families. Affected individuals presented with nail dystrophy, palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, dysphagia, deafness, and asthma. Whole exome sequencing identified homozygous GRHL2 mutations in both families, demonstrating its role in skin development and human disease. Analysis of patient cells showed altered cell morphology, impaired tight junctions, and cytoplasmic translocation of GRHL2, indicating pathogenic effects of the mutations.
GRHL2 mutations were found to cause an autosomal recessive ectodermal dysplasia syndrome in two unrelated Kuwaiti families. Affected individuals presented with nail dystrophy, palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, dysphagia, deafness, and asthma. Whole exome sequencing identified homozygous GRHL2 mutations in both families, demonstrating its role in skin development and human disease. Analysis of patient cells showed altered cell morphology, impaired tight junctions, and cytoplasmic translocation of GRHL2, indicating pathogenic effects of the mutations.
GRHL2 mutations were found to cause an autosomal recessive ectodermal dysplasia syndrome in two unrelated Kuwaiti families. Affected individuals presented with nail dystrophy, palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, dysphagia, deafness, and asthma. Whole exome sequencing identified homozygous GRHL2 mutations in both families, demonstrating its role in skin development and human disease. Analysis of patient cells showed altered cell morphology, impaired tight junctions, and cytoplasmic translocation of GRHL2, indicating pathogenic effects of the mutations.
Mutations in GRHL2 Result in an Autosomal-Recessive
Ectodermal Dysplasia Syndrome Gabriela Petrof, 1 Arti Nanda, 2 Jake Howden, 3 Takuya Takeichi, 1,4 James R. McMillan, 5 Sophia Aristodemou, 5 Linda Ozoemena, 5 Lu Liu, 5 Andrew P. South, 6 Celine Pourreyron, 6 Dimitra Dafou, 7 Laura E. Proudfoot, 1 Hejab Al-Ajmi, 2 Masashi Akiyama, 4 W.H. Irwin McLean, 6 Michael A. Simpson, 8 Maddy Parsons, 3 and John A. McGrath 1,6, * Grainyhead-like 2, encoded by GRHL2, is a member of a highly conserved family of transcription factors that play essential roles during epithelial development. Haploinsufciency for GRHL2 has been implicated in autosomal-dominant deafness, but mutations have not yet been associated with any skin pathology. We investigated two unrelated Kuwaiti families in which a total of six individuals have had lifelong ectodermal defects. The clinical features comprised nail dystrophy or nail loss, marginal palmoplantar keratoderma, hypo- dontia, enamel hypoplasia, oral hyperpigmentation, and dysphagia. In addition, three individuals had sensorineural deafness, and three had bronchial asthma. Taken together, the features were consistent with an unusual autosomal-recessive ectodermal dysplasia syn- drome. Because of consanguinity in both families, we used whole-exome sequencing to search for novel homozygous DNA variants and found GRHL2 mutations common to both families: affected subjects in one family were homozygous for c.1192T>C (p.Tyr398His) in exon 9, and subjects in the other family were homozygous for c.1445T>A (p.Ile482Lys) in exon 11. Immortalized keratinocytes (p.Ile482Lys) showed altered cell morphology, impaired tight junctions, adhesion defects, and cytoplasmic translocation of GRHL2. Whole-skintranscriptomic analysis (p.Ile482Lys) disclosed changes in genes implicated in networks of cell-cell and cell-matrix adhesion. Our clinical ndings of an autosomal-recessive ectodermal dysplasia syndrome provide insight into the role of GRHL2 in skin develop- ment, homeostasis, and human disease. Grainyhead-like 2 (GRHL2) is a mammalian homolog of Drosophila protein grainy head (GRH), which, along with GRHL1 and GRHL3, has a role in epithelial morphogen- esis. 1,2 This family of transcription factors controls the development and differentiation of multicellular epithelia by regulating genes germane to cell junction formation and proliferation. 3,4 Biologically, GRHL2 contributes to formation of the epithelial barrier and wound healing, as well as neural-tube closure, maintenance of the muco- ciliary airway epithelium, and tumor suppression. 511 GRHL2 (MIM 608576) has been shown to regulate TERT (MIM 187270) expression and to enhance proliferation of epidermal keratinocytes; it also impairs keratinocyte dif- ferentiation through transcription inhibition of genes clustered at the epidermal differentiation complex 12 and regulates epithelial morphogenesis by establishing func- tional tight junctions. 13 GRHL2 is also present in the cochlear duct, 14 and mu- tations in human GRHL2 have been found in progres- sive autosomal-dominant hearing loss (DFNA28 [MIM 608641]), 15,16 and other polymorphic sequence variants in GRHL2 have been implicated in age-related hearing impairment and noise-induced hearing loss. 1719 To date, however, the role of GRHL2 in skin biology has not been well established. Causing severe facial and neural-tube de- fects, Grhl2 knockout is embryonically lethal in mice, 17,20 and mutant zebrash display inner-ear defects and abnormal swimming positions. 18 In contrast, Grhl1 / mice show hair loss and palmoplantar keratoderma, as well as abnormal desmosome cell junctions and dysregu- lated terminal differentiation in keratinocytes. 21 More- over, Grhl3 / embryos fail to establish a normal epidermal barrier and display defective embryonic wound repair. 22 Thus, unlike for GRHL1 and GRHL3, there is currently a lack of data associating GRHL2 with skin pathology. In this report, however, we have identied two families in which affected subjects have developmental defects affecting skin, oral mucosa, and teeth (as well as hearing and lungs), thus implicating GRHL2 in an autosomal-reces- sive ectodermal dysplasia syndrome. We investigated two unrelated Kuwaiti families, both consanguineous, in which clinically similar features were present in a total of six affected individuals (Figures 1A and 1B). The clinical features were noted in early infancy and comprised short stature (%25 th percentile), nail dystrophy and/or loss, oral mucosa and/or tongue pigmen- tation, abnormal dentition (delay, hypodontia, enamel hy- poplasia), keratoderma affecting the margins of the palms 1 St. Johns Institute of Dermatology, Kings College London, Guys Campus, London SE1 9RT, UK; 2 Asad Al-Hamad Dermatology Center, Al-Sabah Hospital, Kuwait City 13001, Kuwait; 3 Randall Division of Cell and Molecular Biophysics, Kings College London, Guys Campus, London SE1 9RT, UK; 4 Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan; 5 National Diagnostic Epidermolysis Bullosa Laboratory, Via- path, St. Thomas Hospital, London SE1 7EH, UK; 6 Dermatology and Genetic Medicine, College of Life Sciences and College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee DD1 5EH, UK; 7 Department of Genetics, Development, and Molecular Biology, School of Biology, Aristotle University, Thessaloniki 54124, Greece; 8 Department of Medical and Molecular Genetics, Kings College London School of Medicine and Guys Hospital, London SE1 9RT, UK *Correspondence: john.mcgrath@kcl.ac.uk http://dx.doi.org/10.1016/j.ajhg.2014.08.001. 2014 The Authors This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 308 The American Journal of Human Genetics 95, 308314, September 4, 2014 and soles, and focal hyperkeratosis of the dorsal aspects of the hands and feet (Figures 1C and 1D; Figure S1, available online). No individual showed any wound-healing defect, blistering tendency, hair or sweating abnormalities, or other developmental anomalies. Two affected sisters (ED- 02 VI-3 and VI-5) had dysphagia with evident esophageal strictures. Three individuals (ED-01 IV-4 and IV-5 and ED-02 VI-3) developed sensorineural deafness in early in- fancy, and three others (ED-01 IV-4 and IV-5 and ED-02 IV-2) had bronchial asthma. One individual (ED-01 IV-4) had severe iron-deciency anemia requiring blood transfu- sion. Laboratory tests (full blood count, serum biochem- istry, immunoglobulin levels, and thyroid-function tests) were otherwise within the normal range for all affected individuals. None of the parents had any skin, hair, teeth, nail, or hearing abnormalities. To investigate the etiology of the condition, we rst assessed lesional skin biopsies taken from three affected individuals (ED-01 IV-4 and ED-02 VI-3 and VI-5) by using immunohistochemistry and transmission electron micro- scopy. The subjects legal guardians provided written informed consent according to a protocol approved by the St. Thomas Hospital Ethics Committee (Molecular basis of inherited skin disease: 07/H0802/104). Blood and skin samples (ellipse of skin taken under local anesthesia by 1% lignocaine) were obtained in adherence to the Declaration of Helsinki guidelines. Light microscopy showed mild acanthosis and hyperkeratosis (Figure S2), but transmission electron microscopy of the skin was unre- markableit showed no clear abnormalities in keratino- cytes, hemidesmosomes, or desmosome cell-cell junctions. Likewise, skin immunolabeling using a panel of antibodies Figure 1. Pedigrees and Clinical Features of This Autosomal-Recessive Ectodermal Dysplasia Syndrome (A and B) Two unrelated consanguineous pedigrees with a total of six affected indi- viduals. (C and D) An affected 8-year-old male (from pedigree ED-01) and an affected 12- year-old female (from pedigree ED-02) both show features of tongue hyperpig- mentation, skin thickening around the margins of the palms and soles, hypoplas- tic nger and toe nails, knuckle pads on the ngers, and atrophic wrinkling on the dorsal aspects of the hands and feet. Addi- tional clinical images from other subjects are shown in Figure S1. that target basement membrane extracellular matrix proteins (collagen IV and collagen VII), epidermal-adhe- sion-associated proteins (desmoglein 1, keratin 1, keratin 14, and desmopla- kin), and markers of terminal differ- entiation (laggrin) showed normal intensity and labeling patterns when affected skin from two affected individuals was compared to skin from a normal control subject (Figure S3; see Sup- plemental Data for methods and antibody details). How- ever, we noted increased staining for the proliferation marker Ki-67 in the epidermis (Figure S4). Collectively, the skin-biopsy ndings were not diagnostic for any known inherited skin disease. We then used whole-exome sequencing to identify a candidate gene or genes. We extracted genomic DNA from peripheral blood from two affected individuals (ED- 01 IV-4 and ED-02 VI-3). We performed whole-exome cap- ture by using in-solution hybridization (Agilent All Exon Kit V4) and generated sequencing on the Illumina HiSeq 2000. Resulting reads were aligned to the reference human genome (UCSC Genome Browser hg19, GRCh37) with the Novoalign software package (Novocraft Technologies). Duplicate reads, resulting from PCR clonality or optical duplicates, and reads mapping to multiple locations were excluded from downstream analysis. A summary of exome-coverage data is presented in Table S1. Sixteen pre- viously unreported homozygous mutations were identied (ten in family ED-01 and six in family ED-02). The only gene containing homozygous variants common to both subjects was GRHL2 (Table S2). The respective mutations were c.1192T>C (p.Tyr398His) in exon 9 and c.1445T>A (p.Ile482Lys) (RefSeq NM_024915.3) in exon 11. These mutations were conrmed by Sanger sequencing (Figures 2A and 2B) and were also shown to segregate with the dis- ease phenotype in other affected pedigree members. Both mutations are located in the DNA binding site of GRHL2 (Figure 2C) and are predicted to be probably damaging by PolyPhen-2 analysis (scores 0.984 and 0.994 for The American Journal of Human Genetics 95, 308314, September 4, 2014 309 c.1192T>C and c.1445T>A, respectively). Neither variant has been observed by the 1000 Genomes Project or de- tected in ~1,200 control in-house exomes or in 260 ethni- cally matched control chromosomes. GRHL2 can be detected in the nuclear fraction of normal human keratinocytes in culture (but is subsequently lost in cell senescence). 23 We cultured keratinocytes from two of the skin biopsies (ED-02 VI-3 and ED-02 VI-5) by standard methods and used these cells to examine GRHL2 expres- sion and GRHL2 localization (see Supplemental Data for methods); both were found to be reduced (Figures 2D and 2E). We then isolated primary keratinocytes from one of the affected individuals (ED-02 VI-5) and immortal- ized these cells at passage 1 (see Supplemental Data for methods). The phenotype of these cells was assessed by confocal microscopy. GRHL2 mutant cells showed a less cuboidal, elongated phenotype and failed to form intact cell junctions, as seen in control immortalized keratino- cytes. Notably, there was a reduction in cell membrane labeling for E-cadherin (adherens junctions) and zona- occludens-2 (tight junctions) (Figure S5). GRHL2 stain- ing in control keratinocytes was seen both at cell-cell Figure 2. Autosomal-Recessive Muta- tions in GRHL2 Lead to Reduced Gene Expression and Protein Levels (A and B) Sanger sequencing conrmed the presence of different homozygous missense mutations in GRHL2 in affected subjects from both pedigrees. (C) Schematic representation of the func- tional domains of GRHL2. The recessive missense mutations we identied (top) are located within the DNA binding domain; the previously reported heterozy- gous splice-site or deletion mutations that cause autosomal-dominant deafness are also illustrated (bottom). (D) qPCR for GRHL2 expression in cultured keratinocytes showed reduced expression in two affected subjects from pedigree ED-02 (*p < 0.05 in comparison to control cells). Error bars represent the SD from three independent experiments. (E) Immunoblotting using cultured kerati- nocyte whole-cell lysates revealed mark- edly reduced or undetectable amounts of GRHL2 in these same individuals. contact areas and within the nu- cleus (Figure 3A), whereas in mutant cells, the signal was not at the pe- riphery and instead showed a frag- mented punctate nuclear localization (Figure 3B). To assess the effects of the mutations on keratinocyte cell function, we also performed assays of cell adhesion and de-adhesion (see Supplemental Data for methods). No differences were noted for cell adhe- sion between mutant and control cells (Figure 3C), but mutant cells detached from bronectin much faster than normal human keratinocyte controls after exposure to trypsin (Figure 3D). Next, we assessed the transcriptome prole by using RNA extracted from whole skin from two individuals in pedigree ED-02. RNA from healthy control skin was ob- tained from discarded abdominoplasty tissue from plastic surgeons and used as four pooled samples. RNA extraction was performed with the Ambion mirVana miRNA Isolation kit (Invitrogen) according to the manufacturers instruc- tions. RNA was amplied with the Illumina TotalPrep RNA Amplication Kit, and subsequent gene-expression proling was performed with the Illumina array Hu- manHT-12 v4 Expression BeadChip Kit according to the manufacturers instructions. Gene-expression data were then analyzed with GenomeStudio software (Illumina). A preltering set was determined for signicantly modulated expression (detection p value < 0.01; signal intensity fold change R 2.0) between affected and control skin. A comprehensive functional-enrichment analysis was then performed with (1) the Database for Annotation, Visualiza- tion, and Integrated Discovery (v.6.7), based on the Gene 310 The American Journal of Human Genetics 95, 308314, September 4, 2014 Ontology (GO) database (see Web Resources), and (2) the GeneGo Metacore software (Thomson Reuters), a sys- tems-biology analysis tool based on a curated database of human protein-protein and protein-DNA interactions, transcription factors, signaling, and metabolic pathways. Comparison of the affected individuals skin with the skin of healthy age- and site-matched control individuals identied 1,457 gene transcripts that were signicantly altered: 668 upregulated (R2-fold change) and 789 down- regulated (%0.5-fold change) transcripts for ED-02 VI-5. For ED-02 VI-3, 1,141 gene transcripts were altered: 466 upregulated and 675 downregulated. Of these changes in gene expression, 359 upregulated and 344 downregulated gene transcripts were common to both affected subjects. Evaluation of the changes in gene expression by func- tional-enrichment analysis identied several enriched GO pathways, processes, networks, and disease-associated transcripts, some of which are germane to the known func- tions of GRHL2. The top three upregulated GO pathways were linked to protein-folding maturation, cytoskeleton remodeling, and transcriptional control of lipid biosyn- thesis (involving genes encoding proopiomelanocortins and mitochondrial enzymes involved in metabolic path- ways) (Tables S3S10). Among the most signicantly upregulated GO networks were the signal-transduction pathways and intermediate-lament remodeling (Table S7). Conversely, immune-response signaling; migration- inhibitory-factor-induced cell adhesion, migration, and angiogenesis; and networks of cell-cell and cell-matrix adhesion were downregulated (Table S8). With regard to skin differentiation and barrier forma- tion, selected alterations in gene expression are presented in Table S11. We also veried potential changes by per- forming quantitative PCR (qPCR) with RNA from whole skin of three individuals from the two pedigrees, as well as immortalized keratinocytes and primary broblasts from one affected person (see Supplemental Data for methods and controls). We observed reduced expression of GRHL2 for all templates and a contrasting increase in GRHL1 (MIM 609786) and GRHL3 (MIM 608317) expres- sion: unique and cooperative roles for this transcription factor family have been previously documented. 24 The most marked skin-barrier-associated gene changes were upregulation of aquaporin-encoding genes AQP5 (MIM Figure 3. Impact of GRHL2 Mutations on Keratinocyte Cell Biology (A) Confocal microscopy in normal keratinocytes revealed nuclear, cytoplasmic, and membranous labeling for an antibody raised against GRHL2. (B) In contrast, keratinocytes froman affected subject showed an altered pattern of antibody localizationwithin the nucleus and a lack of any cell membrane labeling. (C) Cell-adhesion assays showed no difference between wild-type and mutant keratinocytes. Error bars represent the SD from three independent experiments. (D) In contrast, mutant cells showed more rapid detachment in trypsin de-adhesion assays. Error bars represent the SD from three independent experiments. NHK stands for normal human keratinocyte. The American Journal of Human Genetics 95, 308314, September 4, 2014 311 600442) and AQP7 (MIM 602974), the latter of which was expressed 5031003 more in affected skin than in control skin. Gain-of-function mutations in AQP5 have previously been associated with a formof autosomal-domi- nant nonepidermolytic palmoplantar keratoderma (MIM 600962). 25 Two-fold or greater reduction in gene expres- sion was noted for S100A8 (MIM 123885) and S100A9 (MIM 123886), known targets for GRHL1. Previously, it has also been shown that GRHL2 enhances skin-barrier function by upregulating the tight-junction components claudins 3 and 4 and also Rab25, which localizes claudin 4 to tight junctions. 26 In affected people, we noted in- creases in CLDN3 (MIM 602910), CLDN4 (MIM 602909), and RAB25 (MIM 612942) expression in whole skin (tran- scriptome and qPCR) and cultured keratinocytes (qPCR). Increased claudin 4 immunolabeling was also noted in the skin of two affected individuals (Figure S5). Labeling for the proliferation marker Ki-67 was increased in the affected subjects skin (Figure S4). This indicates that suprabasal keratinocytes are subject to an abnormal termi- nal-differentiation program, which provides a possible explanation for thickening of the epidermis and impair- ment of the epidermal barrier in our affected individuals with mutant GRHL2, although it is unclear why the most prominent skin scaling was found around the margins of the soles. We also noted reduced expression of TERT in the affected individuals skin and keratinocytes. Over- expression of GRHL2 in normal keratinocytes increases telomerase activity and increases replicative life span (TERT and PCNA [MIM 176740]). In contrast, knockdown of GRHL2 represses the expression of these genes. 12 The impact of GRHL2 mutations on cell morphology has been previously described. 4 Lung epithelial cells trans- duced with Grhl2 small hairpin RNA atten in culture and lose their cuboidal morphology into an expanded cell phenotype. Knocking down Grhl2 in lung epithelial cell lines leads to downregulation of Cldn4 and Cdh1. 9 In our subjects keratinocytes, immunostaining with E-cadherin showed reduced expression and qPCR showed downregu- lation of this transcript (Figure S6). In addition to being expressed in skin, Grhl2 and GRHL2 are highly expressed in the inner ear, the lung epithelium, the ureteric bud of the kidney, the olfactory epithelium, the urogenital tract, the gastric mucosa, and human breast cancer cells. 4,811,18,27,28 With regard to the clinical pheno- type in our affected individuals, aside from the changes affecting the skin and oral mucosa, the other main features comprised deafness and asthma, although this was variably present. Three subjects (ED-01 IV-4 and IV-5 and ED-02 VI-3) had deafness that developed in early in- fancy (c.f. the later-onset deafness in other families with GRHL2 haploinsufciency). 15,16 Of note, none of the het- erozygous carriers of either missense mutation in GRHL2 had any deafness. The signicance of GRHL2 in vertebrate inner-ear development is well established, 16 but the lack of deafness in the heterozygotes (and some homozygotes) in our pedigrees indicates a different functional effect of the missense mutations. Deafness is not a common feature of ectodermal dysplasia syndromes, although hearing loss can result from abnormalities in p63 and Notch signaling (morphological defects in organ of Corti) 29 and mutations in connexins 26 and 30 (altered endolymph ion homeosta- sis). 30 In contrast, mutagenesis studies in Grhl2 have indi- cated a probable different pathophysiology for deafness with enlarged otocysts, absent otoliths, and malformed semicircular canals. 16 The observation that three of the affected individuals (ED-01 IV-4 and IV-5 and ED-02 IV-2) had clinical symp- toms of asthma is also noteworthy because the top en- riched GO disease among the downregulated transcripts in our microarray data was asthma (Table S10). Previous in situ hybridization analyses have indicated that Grhl2 is the only family member that is highly expressed in distal lung epithelium throughout development, although the particular cells expressing Grhl2 have not been identied, nor has its functional role in the lung epithelium been fully established. 4 Grhl1 and Grhl3, in contrast, are ex- pressed in the embryonic lung epithelium, but later their expression is reduced in bronchi and bronchioles and is undetectable in the alveolar lung epithelium. 4,27 The potential relevance of other sequence variants in GRHL2 to sporadic or familial cases of human asthma and other obstructive-airway diseases remains to be determined. In summary, GRHL2, a member of a family of highly conserved transcription factors, is implicated in epithelial morphogenesis across a number of species. We have used whole-exome sequencing to identify GRHL2 mutations underlying an ectodermal dysplasia syndrome in two fam- ilies, and our data expand the current knowledge about the role of GRHL2 in human disease and epithelial cell biology. Supplemental Data Supplemental Data include 6 gures and 13 tables and can be found with this article online at http://dx.doi.org/10.1016/j. ajhg.2014.08.001. Acknowledgments The Centre for Dermatology and Genetic Medicine is supported by a Wellcome Trust Strategic Award (reference 098439/Z/12/Z). This work was supported by the Biotechnology and Biological Sciences Research Council, the Royal Society, and the UK National Institute for Health Research comprehensive Biomedical Research Centre award to Guys and St. Thomas NHS Foundation Trust in partner- ship with the Kings College London and Kings College Hospital NHS Foundation Trust. This study was also supported, in part, by DebRA UK, the Great Britain Sasakawa Foundation (award 4314), and the Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation (S2404) from the Japan Society for the Promotion of Science. We also thank Venu Pullabhatla for assistance with transcriptomic data analysis and access. Received: June 11, 2014 Accepted: August 1, 2014 Published: August 21, 2014 312 The American Journal of Human Genetics 95, 308314, September 4, 2014 Web Resources The URLs for data presented herein are as follows: Ensembl Genome Browser, http://www.ensembl.org/index.html Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/ Gene Ontology Consortium, http://www.geneontology.org/ Online Mendelian Inheritance in Man (OMIM), http://www. omim.org/ Primer3, http://frodo.wi.mit.edu/primer3/ PubMed, http://www.ncbi.nlm.nih.gov/PubMed/ RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq UCSC Genome Browser, http://genome.ucsc.edu/ Accession Numbers The Gene Expression Omnibus accession number for the micro- array data reported in this paper is number GSE56486. References 1. Venkatesan, K., McManus, H.R., Mello, C.C., Smith, T.F., and Hansen, U. (2003). Functional conservation between mem- bers of an ancient duplicated transcription factor family, LSF/Grainyhead. Nucleic Acids Res. 31, 43044316. 2. Stramer, B., and Martin, P. (2005). Cell biology: master regula- tors of sealing and healing. Curr. Biol. 15, R425R427. 3. Werth, M., Walentin, K., Aue, A., Schonheit, J., Wuebken, A., Pode-Shakked, N., Vilianovitch, L., Erdmann, B., Dekel, B., Bader, M., et al. (2010). The transcription factor grainyhead- like 2 regulates the molecular composition of the epithelial apical junctional complex. Development 137, 38353845. 4. Varma, S., Cao, Y., Tagne, J.B., Lakshminarayanan, M., Li, J., Friedman, T.B., Morell, R.J., Warburton, D., Kotton, D.N., and Ramirez, M.I. (2012). The transcription factors Grainy- head-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differ- entiation. J. Biol. Chem. 287, 3728237295. 5. Bray, S.J., and Kafatos, F.C. (1991). Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila. Genes Dev. 5, 16721683. 6. Narasimha, M., Uv, A., Krejci, A., Brown, N.H., and Bray, S.J. (2008). Grainy head promotes expression of septate junction proteins and inuences epithelial morphogenesis. J. Cell Sci. 121, 747752. 7. Cieply, B., Farris, J., Denvir, J., Ford, H.L., and Frisch, S.M. (2013). Epithelial-mesenchymal transition and tumor sup- pression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2. Cancer Res. 73, 62996309. 8. Werner, S., Frey, S., Riethdorf, S., Schulze, C., Alawi, M., Kling, L., Vafaizadeh, V., Sauter, G., Terracciano, L., Schumacher, U., et al. (2013). Dual roles of the transcription factor grainyhead- like 2 (GRHL2) in breast cancer. J. Biol. Chem. 288, 22993 23008. 9. Varma, S., Mahavadi, P., Sasikumar, S., Cushing, L., Hyland, T., Rosser, A.E., Riccardi, D., Lu, J., Kalin, T.V., Kalinichenko, V.V., et al. (2014). Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idio- pathic pulmonary brosis and mouse models of pulmonary brosis. Am. J. Physiol. LungCell. Mol. Physiol. 306, L405L419. 10. Mlacki, M., Darido, C., Jane, S.M., and Wilanowski, T. (2014). Loss of Grainy head-like 1 is associated with disruption of the epidermal barrier and squamous cell carcinoma of the skin. PLoS ONE 9, e89247. 11. Xiang, J., Fu, X., Ran, W., Chen, X., Hang, Z., Mao, H., and Wang, Z. (2013). Expression and role of grainyhead-like 2 in gastric cancer. Med. Oncol. 30, 714. 12. Chen, W., Xiao Liu, Z., Oh, J.E., Shin, K.H., Kim, R.H., Jiang, M., Park, N.H., and Kang, M.K. (2012). Grainyhead-like 2 (GRHL2) inhibits keratinocyte differentiation through epige- netic mechanism. Cell Death Dis. 3, e450. 13. Senga, K., Mostov, K.E., Mitaka, T., Miyajima, A., and Tani- mizu, N. (2012). Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among clau- din3, claudin4, and Rab25. Mol. Biol. Cell 23, 28452855. 14. Wilanowski, T., Tuckeld, A., Cerruti, L., OConnell, S., Saint, R., Parekh, V., Tao, J., Cunningham, J.M., and Jane, S.M. (2002). A highly conserved novel family of mammalian devel- opmental transcription factors related to Drosophila grainy- head. Mech. Dev. 114, 3750. 15. Peters, L.M., Anderson, D.W., Grifth, A.J., Grundfast, K.M., San Agustin, T.B., Madeo, A.C., Friedman, T.B., and Morell, R.J. (2002). Mutation of a transcription factor, TFCP2L3, causes progressive autosomal dominant hearing loss, DFNA28. Hum. Mol. Genet. 11, 28772885. 16. Vona, B., Nanda, I., Neuner, C., Muller, T., and Haaf, T. (2013). Conrmation of GRHL2 as the gene for the DFNA28 locus. Am. J. Med. Genet. A. 161A, 20602065. 17. Van Laer, L., Van Eyken, E., Fransen, E., Huyghe, J.R., Topsa- kal, V., Hendrickx, J.J., Hannula, S., Maki-Torkko, E., Jensen, M., Demeester, K., et al. (2008). The grainyhead like 2 gene (GRHL2), alias TFCP2L3, is associated with age-related hearing impairment. Hum. Mol. Genet. 17, 159169. 18. Han, Y., Mu, Y., Li, X., Xu, P., Tong, J., Liu, Z., Ma, T., Zeng, G., Yang, S., Du, J., and Meng, A. (2011). Grhl2 deciency impairs otic development and hearing ability in a zebrash model of the progressive dominant hearing loss DFNA28. Hum. Mol. Genet. 20, 32133226. 19. Li, X., Huo, X., Liu, K., Li, X., Wang, M., Chu, H., Hu, F., Sheng, H., Zhang, Z., and Zhu, B. (2013). Association between genetic variations in GRHL2 and noise-induced hearing loss in Chinese high intensity noise exposed workers: a case-con- trol analysis. Ind. Health 51, 612621. 20. Rifat, Y., Parekh, V., Wilanowski, T., Hislop, N.R., Auden, A., Ting, S.B., Cunningham, J.M., and Jane, S.M. (2010). Regional neural tube closure dened by the Grainy head-like transcrip- tion factors. Dev. Biol. 345, 237245. 21. Wilanowski, T., Caddy, J., Ting, S.B., Hislop, N.R., Cerruti, L., Auden, A., Zhao, L.L., Asquith, S., Ellis, S., Sinclair, R., et al. (2008). Perturbed desmosomal cadherin expression in grainy head-like 1-null mice. EMBO J. 27, 886897. 22. Ting, S.B., Caddy, J., Hislop, N., Wilanowski, T., Auden, A., Zhao, L.L., Ellis, S., Kaur, P., Uchida, Y., Holleran, W.M., et al. (2005). A homolog of Drosophila grainy head is essential for epidermal integrity in mice. Science 308, 411413. 23. Chen, W., Dong, Q., Shin, K.H., Kim, R.H., Oh, J.E., Park, N.H., and Kang, M.K. (2010). Grainyhead-like 2 enhances the human telomerase reverse transcriptase gene expression by inhibiting DNA methylation at the 5 0 -CpG island in normal human keratinocytes. J. Biol. Chem. 285, 4085240863. 24. Boglev, Y., Wilanowski, T., Caddy, J., Parekh, V., Auden, A., Darido, C., Hislop, N.R., Cangkrama, M., Ting, S.B., and Jane, S.M. (2011). The unique and cooperative roles of the Grainy The American Journal of Human Genetics 95, 308314, September 4, 2014 313 head-like transcriptionfactors inepidermal development reect unexpected target gene specicity. Dev. Biol. 349, 512522. 25. Blaydon, D.C., Lind, L.K., Plagnol, V., Linton, K.J., Smith, F.J., Wilson, N.J., McLean, W.H., Munro, C.S., South, A.P., Leigh, I.M., et al. (2013). Mutations inAQP5, encodingawater-channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma. Am. J. Hum. Genet. 93, 330335. 26. Tanimizu, N., and Mitaka, T. (2013). Role of grainyhead-like 2 in the formation of functional tight junctions. Tissue Barriers 1, e23495. 27. Kang, X., Chen, W., Kim, R.H., Kang, M.K., and Park, N.H. (2009). Regulation of the hTERT promoter activity by MSH2, the hnRNPs K and D, and GRHL2 in human oral squamous cell carcinoma cells. Oncogene 28, 565574. 28. Auden, A., Caddy, J., Wilanowski, T., Ting, S.B., Cunningham, J.M., and Jane, S.M. (2006). Spatial and temporal expression of the Grainyhead-like transcription factor family during murine development. Gene Expr. Patterns 6, 964970. 29. Terrinoni, A., Serra, V., Bruno, E., Strasser, A., Valente, E., Flores, E.R., van Bokhoven, H., Lu, X., Knight, R.A., and Melino, G. (2013). Role of p63 and the Notch pathway in co- chlea development and sensorineural deafness. Proc. Natl. Acad. Sci. USA 110, 73007305. 30. Terrinoni, A., Codispoti, A., Serra, V., Bruno, E., Didona, B., Paradisi, M., Nistico` , S., Campione, E., Napolitano, B., Diluvio, L., and Melino, G. (2010). Connexin 26 (GJB2) mutations as a cause of the KID syndrome with hearing loss. Biochem. Biophys. Res. Commun. 395, 2530. 314 The American Journal of Human Genetics 95, 308314, September 4, 2014