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Abnormal Cornified Cell Envelope Formation in Mutilating Palmoplantar Keratoderma Unrelated to Epidermal Differentiation Complex

1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.1998.00230.x

1523-1747

Masashi Akiyama, Angela M. Christiano, Kozo Yoneda, Hiroshi Shimizu,

Contact Dermatitis and Allergies

Mutilating palmoplantar keratoderma represents a heterogeneous group of disorders, unified by characteristic mutilation of the fingers or toes, associated with palmoplantar keratoderma. Although loricrin gene mutations were recently reported in Vohwinkel's syndrome and erythrokeratoderma, the genetic basis of mutilating palmoplantar keratoderma is largely unexplored. We studied a family of non-Vohwinkel's syndrome, nonerythrokeratoderma mutilating palmoplantar keratoderma. The proband and his sister were similarly affected. Recessive inheritance was expected from the consanguineous family history. The patients had hyperkeratosis restricted to the palms and the soles. No other body sites were affected. Digital constriction was seen on all the fingers and the mutilation was severe on the distal interphalangeal region of several fingers. Histopathologically, hyperkeratosis without parakeratosis was seen in the lesional skin. Ultrastructural, immunohistochemical, and immunoelectron microscopic analyses revealed malformed cornified cell envelopes, the abnormal intracytoplasmic loricrin retention, and reduced deposition of loricrin to cornified cell envelopes. Involucrin and small proline-rich proteins 1 and 2 were normally distributed. Sequencing of the entire exons and exon-intron borders of loricrin gene of the patients excluded a mutation in loricrin DNA sequence. Linkage analysis excluded the possibility of causative mutation in the epidermal differentiation complex region of 1q21, including loricrin, involucrin, small proline-rich proteins, filaggrin, and trichohyalin. These data confirm the presence of non-Vohwinkel's syndrome mutilating palmoplantar keratoderma phenotype with abnormal loricrin cross-linking at the final stage of cornified cell envelope formation, which is caused by mutations outside the epidermal differentiation complex region. Mutilating palmoplantar keratoderma represents a heterogeneous group of disorders, unified by characteristic mutilation of the fingers or toes, associated with palmoplantar keratoderma. Although loricrin gene mutations were recently reported in Vohwinkel's syndrome and erythrokeratoderma, the genetic basis of mutilating palmoplantar keratoderma is largely unexplored. We studied a family of non-Vohwinkel's syndrome, nonerythrokeratoderma mutilating palmoplantar keratoderma. The proband and his sister were similarly affected. Recessive inheritance was expected from the consanguineous family history. The patients had hyperkeratosis restricted to the palms and the soles. No other body sites were affected. Digital constriction was seen on all the fingers and the mutilation was severe on the distal interphalangeal region of several fingers. Histopathologically, hyperkeratosis without parakeratosis was seen in the lesional skin. Ultrastructural, immunohistochemical, and immunoelectron microscopic analyses revealed malformed cornified cell envelopes, the abnormal intracytoplasmic loricrin retention, and reduced deposition of loricrin to cornified cell envelopes. Involucrin and small proline-rich proteins 1 and 2 were normally distributed. Sequencing of the entire exons and exon-intron borders of loricrin gene of the patients excluded a mutation in loricrin DNA sequence. Linkage analysis excluded the possibility of causative mutation in the epidermal differentiation complex region of 1q21, including loricrin, involucrin, small proline-rich proteins, filaggrin, and trichohyalin. These data confirm the presence of non-Vohwinkel's syndrome mutilating palmoplantar keratoderma phenotype with abnormal loricrin cross-linking at the final stage of cornified cell envelope formation, which is caused by mutations outside the epidermal differentiation complex region. cornified cell envelope epidermal differentiation complex erythrokeratoderma mutilating palmoplantar keratoderma palmoplantar keratoderma small proline-rich protein Vohwinkel's syndrome One of the striking events during the process of the terminal differentiation in stratified squamous epithelia such as the epidermis is the formation of a 15 nm thick layer of protein on the inner surface of the cell periphery, termed the cornified cell envelope (CCE) (Hohl, 1990Hohl D. Cornified cell envelope.Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (164) Google Scholar;Greenberg et al., 1991Greenberg C.S. Birckbichler P.J. Rice R.H. Transglutaminases: multifunctional cross-linking enzyme that stabilizes tissues.Faseb J. 1991; 5: 3071-3077Crossref PubMed Scopus (913) Google Scholar;Reichert et al., 1993Reichert U. Michel S. Schmidt R. The cornified envelope: a key structure of terminally differentiated keratinocytes.in: Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press, San Diego1993: 107-150Google Scholar). The CCE is now known to be assembled by the accumulation of several distinct proteins, including involucrin (Rice and Green, 1979Rice R.H. Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (620) Google Scholar;Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin – structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar), cystatin a (previously known as keratolinin) (Zettergren et al., 1984Zettergren J.G. Peterson L.I. Wuepper K.D. Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue.Proc Natl Acad Sci USA. 1984; 81: 238-242Crossref PubMed Scopus (77) Google Scholar;Kartasova et al., 1987Kartasova T. Cornelissen B.J.C. van der Putte P. Effects of UV, 4-NQO and TPA on gene expression in cultured human keratinocytes.Nucl Acids Res. 1987; 15: 5945-5962Crossref PubMed Scopus (80) Google Scholar;Takahashi et al., 1992Takahashi M. Tezuka T. Katunuma N. Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified cell envelope.FEBS Lett. 1992; 308: 79-82Abstract Full Text PDF PubMed Scopus (69) Google Scholar), several small proline-rich proteins (SPRP) (Kartasova et al., 1987Kartasova T. Cornelissen B.J.C. van der Putte P. Effects of UV, 4-NQO and TPA on gene expression in cultured human keratinocytes.Nucl Acids Res. 1987; 15: 5945-5962Crossref PubMed Scopus (80) Google Scholar;Backendorf and Hohl, 1992Backendorf C. Hohl D.A. A common origin for cornified cell envelope proteins?.Nature Genet. 1992; 2: 91Crossref PubMed Scopus (87) Google Scholar;Gibbs et al., 1993Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. de Putte P. Backendorf C. Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins.Genomics. 1993; 16: 630-637Crossref PubMed Scopus (174) Google Scholar), trichohyalin (Lee et al., 1993Lee S.C. Kim I.G. Marekov L.N. O'keefe E.J. Parry D.A. Steinert P.M. The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar), loricrin (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (360) Google Scholar;Hohl et al., 1991Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.J Biol Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar;Yoneda et al., 1992Yoneda K. Hohl D. McBride O.W. Idler W. Wang M. Cehrs K.U. Steinert P.M. The human loricrin gene.J Biol Chem. 1992; 267: 18060-18066Abstract Full Text PDF PubMed Google Scholar;Yoneda and Steinert, 1993Yoneda K. Steinert P.M. Overexpression of human loricrin in transgenic mice produces a normal phenotype.Proc Natl Acad Sci USA. 1993; 90: 10754-10758Crossref PubMed Scopus (56) Google Scholar), and possibly filaggrin (Richards et al., 1988Richards S. Scott I.R. Harding C.R. Liddell J.E. Powell G.M. Curtis C.G. Evidence for filaggrin as a component of the cell envelope of the newborn rat.Biochem J. 1988; 253: 153-160Crossref PubMed Scopus (41) Google Scholar;Steven and Steinert, 1994Steven A.C. Steinert P.M. Protein composition of the cornified cell envelope of epidermal keratinocytes.J Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar) and keratin intermediate filaments (Abernethy et al., 1977Abernethy J.L. Hill R.L. Goldsmith L.A. Ipsilon-(gamma-glutamyl) lysine cross-links in human stratum corneum.J Cell Biol. 1977; 252: 1837-1839Google Scholar;Steven and Steinert, 1994Steven A.C. Steinert P.M. Protein composition of the cornified cell envelope of epidermal keratinocytes.J Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar). Among these molecules, loricrin is one of the major precursor proteins of CCE (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (460) Google Scholar). A clinically and genetically heterogeneous group of disorders, known collectively as the palmoplantar keratodermas (PPK), are unified by the phenotypic characteristic thickening of the skin over the palms and soles (Stevens et al., 1996Stevens H.P. Kelsell D.P. Bryant S.P. et al.Linkage of an American pedigree with palmoplantar keratoderma and malignancy (palmoplantar ectodermal dysplasia type III) to 17q24. Literature survey and proposed updated classification of the keratodermas.Arch Dermatol. 1996; 132: 640-651Crossref PubMed Google Scholar). Although spectacular progress has been made in the molecular basis of inherited disorders of the skin in recent years, the genetic defects underlying the PPK are still largely unknown. Epidermolytic PPK is caused by mutations in keratin 9 (Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (779) Google Scholar;Bonifas et al., 1994Bonifas J.M. Matsumura K. Chen M.A. et al.Mutations of keratin 9 in two families with palmoplantar epidermolytic hyperkeratosis.J Invest Dermatol. 1994; 103: 474-477Crossref PubMed Scopus (68) Google Scholar;Rothnagel et al., 1995Rothnagel J.A. Wojcik S. Liefer K.M. Dominey A.M. Huber M. Hohl D. Roop D.R. Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma.J Invest Dermatol. 1995; 104: 430-433Crossref PubMed Scopus (60) Google Scholar;Navsaria et al., 1995Navsaria H.A. Swensson O. Ratnavel R.C. et al.Ultrastructural changes resulting from keratin 9 gene mutations in two families with epidermolytic palmoplantar keratoderma.J Invest Dermatol. 1995; 104: 425-429Crossref PubMed Scopus (51) Google Scholar), which is expressed exclusively in the skin of palms and soles. In contrast, less is known about the molecular pathology of nonepidermolytic PPK. There have been reports of linkage of the diffuse nonepidermolytic PPK to keratin gene clusters on both chromosome 17q (Rogaev et al., 1993Rogaev E.I. Rogaeva E.A. Ginter E.K. et al.Identification of the genetic locus for keratosis palmaris et plantaris on chromosome 17 near the RARA and keratin type 1 genes.Nature Genet. 1993; 5: 158-162Crossref PubMed Scopus (32) Google Scholar) and chromosome 12q (Lind et al., 1994Lind L. Lundström A. Hofer P. Holmgren G. The gene for diffuse palmoplantar keratoderma of the type found in northern Sweden is localized to chromosome 12q11-q13.Hum Mol Genet. 1994; 3: 1789-1793Crossref PubMed Scopus (40) Google Scholar) and a mutation in the V1 domain of keratin 1 was also reported in a diffuse nonepidermolytic PPK (Kimonis et al., 1994Kimonis V. DiGiovanna J.J. Yang J.M. Doyle S.Z. Bale S.J. Compton J.G. A mutation in the V1 end domain of keratin 1 in non-epidermolytic palmar-plantar keratoderma.J Invest Dermatol. 1994; 103: 764-769Crossref PubMed Scopus (128) Google Scholar). Keratin 16 mutations have been reported in focal nonepidermolytic PPK (Shamsher et al., 1995Shamsher M.K. Navsaria H.A. Stevens H.P. et al.Novel mutations in keratin 16 gene underlie focal non-epidermolytic palmoplantar keratoderma (NEPPK) in two families.Hum Mol Genet. 1995; 4: 1875-1881Crossref PubMed Scopus (107) Google Scholar), and mutations in keratin 6, 16, and 17 in the related disorder, pachyonychia congenita (Bowden et al., 1995Bowden P.E. Haley J.L. Kansky A. Rothnagel J.A. Jones D.O. Turner R.J. Mutation of a type II keratin gene (K6a) in pachyonychia congenita.Nature Genet. 1995; 10: 363-365Crossref PubMed Scopus (204) Google Scholar;McLean et al., 1995McLean W.H.I. Rugg E.L. Lunny D.P. et al.Keratin 16 and keratin 17 mutations cause pachyonychia congenita.Nature Genet. 1995; 9: 273-278Crossref PubMed Scopus (279) Google Scholar). There has only been one report of linkage of a PPK, which maps to chromosome 18q near the desmosomal cadherin gene cluster (Hennies et al., 1995Hennies H.C. Kuester W. Mischke D. Reis A. Localization of a locus for the striated form palmoplantar keratoderma to chromosome 18q near the desmosomal cadherin gene cluster.Hum Mol Genet. 1995; 4: 1015-1020Crossref PubMed Scopus (61) Google Scholar). In spite of these reports, we cannot exclude possible candidate molecules for other phenotypes of PPK (Magro et al., 1997Magro C.M. Baden L.A. Crowson A.N. Bowden P.E. Baden H.P. A novel nonepidermolytic palmoplantar keratoderma: a clinical and histopathologic study of six cases.J Am Acad Dermatol. 1997; 37: 27-33Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Recently, loricrin gene mutations were reported as causative defects in two distinct types of PPK, Vohwinkel's syndrome (VS) associated with ichthyosis (Maestrini et al., 1996Maestrini E. Monaco A.P. McGrath J.A. et al.A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel's syndrome.Nature Genet. 1996; 13: 70-77Crossref PubMed Scopus (199) Google Scholar;Korge et al., 1997Korge B.P. Ishida-yamamoto A. Pünter C. et al.Loricrin mutation in Vohwinkel's keratoderma is unique to the variant with ichthyosis.J Invest Dermatol. 1997; 109: 604-610Abstract Full Text PDF PubMed Scopus (93) Google Scholar) and erythrokeratoderma (EK) (Ishida-yamamoto et al., 1997Ishida-yamamoto A. McGrath J.A. Lam H.-M. Iizuka H. Christiano A.M. The molecular basis of autosomal dominant erythrokeratoderma: a frameshift mutation in the loricrin gene.Am J Hum Genet. 1997; 61: 581-589Abstract Full Text PDF PubMed Scopus (118) Google Scholar). Mutilation of the fingers is a characteristic clinical feature shared by these two types of PPK. In this context, one can predict that other forms of PPK with mutilation might be due to alterations in CCE formation. These facts stimulated us to study CCE formation, especially loricrin, in a family of non-VS, non-EK mutilating PPK (MPPK) in order to elucidate whether a loricrin abnormality is a common cause of mutilating phenotype of PPK. The present ultrastructural, immunofluorescent, and immunoelectron microscopic observations and DNA analysis suggest that abnormal loricrin cross-linking in the formation of CCE also underlies the phenotype of non-VS, non-EK MPPK in which loricrin gene mutations are absent. The proband, also referred to as case 1, was a 38 y old male of Japanese origin, who was the first child of consanguineous parents (Figure 1a). His maternal grandmother and paternal grandmother are sisters. His sister (case 2) was similarly affected with MPPK. There is no other patient with MPPK in the family as far as we know. Case 1 presented with palmoplantar keratotic lesions that developed from his infancy (Figure 2a), gradually progressed, and became stable in adolescence. He had no other skin abnormalities. His hair, teeth, and nails appeared normal. His hearing was normal and he had no other congenital anomalies. His palms and soles were hyperkeratotic and had a waxy appearance. Pseudoainhum was noted on the joints of his right fourth and fifth and left third fingers, and mild constrictions were rotated on the joints of other fingers (Figure 2a). Surgical excision of the constricted fourth finger and skin graft on the DIP joint of the right middle finger was carried out about 10 y ago. Skin biopsies were obtained from an involved, constricted area of the second finger and an uninvolved area of the forearm area under local anesthesia after informed consent, and were processed for conventional light and electron microscopy, immunohistology, and immunoelectron microscopy.Figure 2Characteristic clinical features and histopathology of the finger lesion of the proband. (a) The palm has a hyperkeratotic waxy appearance. Marked pseudoainhum is noted on the DIP joint of the right fourth and fifth and left third fingers. (b) Marked hyperkeratosis and moderate acanthosis are seen (hematoxylin and eosin stain). Hypergranulosis is present but parakeratosis is not seen. Scale bar, 100 μm.View Large Image Figure ViewerDownload (PPT) Case 2, the 33 y old sister of the proband, had very similar lesions to case 1. She displayed well demarcated hyperkeratosis on the palms and the soles with constrictions on the DIP joints of the fingers. Her hair, teeth, and nails appeared normal. Her hearing was normal as well. The primary antibodies to CCE precursor proteins used in this study were rabbit polyclonal anti-human involucrin antibody (Biomedical Technologies, Stoughton, MA), mouse monoclonal anti-human SPRP 1 and 2 antibody (Kim et al., 1995Kim S.-Y. Chung S.-I. Yoneda K. Steinert P.M. Expression of transglutaminase 1 in human epidermis.J Invest Dermatol. 1995; 104: 211-217Crossref PubMed Scopus (90) Google Scholar), and rabbit polyclonal anti-human loricrin antibody (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (360) Google Scholar). Six micrometer thick sections of fresh skin cut by cryostat were used as substrate. The sections were incubated in normal horse and goat sera for 30 min, and then incubated in primary antibody solution for 1 h at 37°C, followed by fluorescein isothiocyanate-conjugated to horse anti-mouse IgG and IgM (Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Sections were extensively washed with phosphate-buffered saline between incubations. Counterstaining was done by incubating the sections in 10 μg propidium iodide per ml (to demonstrate nuclei) (Sigma, St. Louis, MO) for 10 s. Stained sections were mounted with a cover slip in mounting medium. Post-embedding immunoelectron microscopy using cryofixed and cryosubstituted skin specimens was carried out as described previously (Shimizu et al., 1989Shimizu H. McDonald J.N. Kennedy A.R. Eady R.A. Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy.Arch Dermatol Res. 1989; 281: 443-448Crossref PubMed Scopus (113) Google Scholar) with slight modification. Briefly, skin specimens were cryofixed by plunging them into liquid propane cooled to –190°C, followed by cryosubstitution; they were then embedded in Lowicryl K11 M (Chemische Werke Lowi, Waldkraiburg, Germany) at –60°C. The specimens were polymerized by ultraviolet irradiation. Ultrathin sections were incubated for 2 h at 37°C with a primary antibody. After being washed, each section was placed on a drop of 1 nm gold-labeled goat anti-mouse or rabbit IgG (Amersham International, Buckinghamshire, U.K.) diluted 1:40 at room temperature for 2 h, and was then washed with distilled water. For easier observation, the 1 nm gold particles were enlarged by incubation with immunogold silver-enhancement solution (Amersham International) at room temperature for 6 min (for observation at high magnification) or 10 min (for observation at low magnification) (Shimizu et al., 1992Shimizu H. Ishida-yamamoto A. Eady R.A. The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.J Histochem Cytochem. 1992; 40: 883-888Crossref PubMed Scopus (49) Google Scholar). The sections were counterstained with saturated uranyl acetate and lead citrate for 6 and 2 min, respectively. DNA was isolated from peripheral blood leukocytes of the proband (case 1), his sister (case 2), and their parents. The entire coding sequence of the loricrin gene was polymerase chain reaction (PCR) amplified from genomic DNA using the kit Expand Long Template PCR System with High Fidelity Taq (Boehringer, Mannheim, Germany), which alleviated the problems due to very high G-C content of the sequence. The primers used to amplify exon 1 of loricrin were 5′-GTTTGACTCTCTTAGGGCAC-3′ (forward) and 5′-GTCAGGCCTGGGCAAGACCA-3′ (reverse), derived from GenBank file M94077, and PCR was carried out at 95°C for 5 min, then 40 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min. The primers used to amplify exon 2, designed from the published sequence (GenBank M94077), were 5′-GCTGAGGCTCTGGCACCTGAAAG-3′ (forward) and 5′-GCCGGAGAGCTCAATGGCTTCT-3′ (reverse), producing a PCR product of 1235 bp. PCR conditions were: 5 min at 95°C, then 1 min at 95°C, 45 s at 65°C, and 2 min at 72°C for 35 cycles. PCR products were verified on agarose gel, purified using the Centriflex Gel Filtration Column from ACGT, and directly sequenced, using the ABI PRISM Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase, FS (Perkin Elmer-ABI, Foster City, CA). The sequences were run on an ABI 310 Prism Sequencer (Applied Biosystems, Foster City, CA). Microsatellite markers used for exclusion of the epidermal differentiation complex (EDC) region were part of the U.K. MRC Human Genome Mapping set (Reed et al., 1994Reed P.W. Davies J.L. Copeman J.B. et al.Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping.Nature Genet. 1994; 7: 390-395Crossref PubMed Scopus (300) Google Scholar). PCR reactions were performed using primers end-labeled with [γ33P]dATP and PCR was carried out according to the following program: 95°C for 5 min, followed by 27 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, and electrophoresis on a 6% acrylamide gel followed by autoradiography. Genotypers were assigned by visual inspection. The marker map used was D1S534–1.0-D1S422–2.2-AFM135yc3–0.1-D1S2345–3.0-D1S2346–1.0-AFMa357ze5–0.1-D1S305–5.4-SPTA1–6.8-D1S484, with distances in centimorgans (Maestrini et al., 1996Maestrini E. Monaco A.P. McGrath J.A. et al.A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel's syndrome.Nature Genet. 1996; 13: 70-77Crossref PubMed Scopus (199) Google Scholar). The order of markers and distances were based on the Généthon (Dib et al., 1996Dib C. Faure S. Fizames C. et al.A comprehensive genetic map of the human genome based on 5,264 microsatellites.Nature. 1996; 380: 152-154Crossref PubMed Scopus (2665) Google Scholar) and the CHLC (Murray et al., 1994Murray J.C. Buetow K.H. Weber J.L. et al.A comprehensive human linkage map with centimorgan density.Science. 1994; 265: 2049-2054Crossref PubMed Scopus (494) Google Scholar) human linkage map. Light microscopically, the biopsy skin sample from an involved area of the finger of the proband showed marked hyperkeratosis, slightly thickened granular cell layer, and mild acanthosis without parakeratosis (Figure 2b). Electron microscopically, basal and spinous cells in the epidermis did not show significant morphologic abnormalities. Keratohyalin granules in the thickened granular cell layers appeared normal (Figure 3a). The transitional cell layer, which is only occasionally observed in normal skin, was frequently encountered between the granular and cornified cells. Thin, electron dense cell envelopes were seen in the superficial granular cells (Figure 3b); however, the increase in thickness of cell envelopes in the horny layer that always occurs in normal skin was not observed (Figure 3c). Lipid droplets and remnants of cellular organelles were present in the cornified cells (Figure 3c). Ring-shaped structures made up of cell membrane with thin CCE and desmosomes were frequently observed in the cytoplasm of the upper granular cells, suggesting phagocytosis or invagination of cell membrane with abnormally formed CCE into the cytoplasm (Figure 3e). Non-desmosomal areas of these ring-shaped structures were very sparsely labeled with loricrin and involucrin antibodies in immunoelectron microscopy (data not shown). Immunofluorescence study revealed an abnormal distribution of loricrin in lesional skin. Membranous loricrin staining was absent in the granular and cornified cells, and a weak cytoplasmic staining was observed in the granular cells (Figure 4a). In the uninvolved site of the patient, loricrin staining was as strong as that in the normal control skin, and its membranous staining can be seen in the granular and upper spinous layers (Figure 4b). In normal control skin, loricrin staining was observed only in the granular layer. In contrast, the expression of other CCE precursor molecules, involucrin, and SPRP 1 and 2, was stronger than that in the normal control skin, and their membranous staining were observed in the upper spinous layer cells as well as granular cells (Figure 5).Figure 5Membranous stainings in the granular and upper spinous layers both for involucrin and for SPRP 1 and 2 are similarly observed in lesional and nonlesional skin of the patient. Involucrin and SPRP 1 and 2 immunolabelings are detected by fluorescein isothiocyanate-conjugated secondary antibody (green) and nuclear stain is carried out by propidium iodide (red). Involucrin immunolabeling in lesional skin (a) and nonlesional skin (b), SPRP 1 and 2 immunolocalization in lesional skin (c) and nonlesional skin (d) of the patient and normal control skin (e). Scale bars, 50 μm.View Large Image Figure ViewerDownload (PPT) By immunoelectron microscopy, CCE were sparsely labeled with the loricrin antibody in the patient's skin (Figure 6a), whereas intense labeling was seen in normal skin (Figure 6b). In addition, immunoelectron microscopy revealed that involucrin labels on CCE in patients skin was rich compared with that in normal skin (Figure 6c, d). Direct sequencing of the loricrin gene of the genomic DNA from the proband (case 1), his affected sister (case 2), and their parents revealed no mutation in both exons and exon-intron borders of the loricrin gene. Assuming an autosomal recessive mode of inheritance, by linkage analysis of three microsatellite markers spanning in 1q21 (D1S2344, D1S2345, D1S305), using DNA samples isolated from the proband, his affected sister, and their parents, the entire EDC region of 1q21 was excluded (Figure 1b). Loricrin is the major protein component of the CCE (Hohl et al., 1991Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.J Biol Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar;Yoneda et al., 1992Yoneda K. Hohl D. McBride O.W. Idler W. Wang M. Cehrs K.U. Steinert P.M. The human loricrin gene.J Biol Chem. 1992; 267: 18060-18066Abstract Full Text PDF PubMed Google Scholar;Yoneda and Steinert, 1993Yoneda K. Steinert P.M. Overexpression of human loricrin in transgenic mice produces a normal phenotype.Proc Natl Acad Sci USA. 1993; 90: 10754-10758Crossref PubMed Scopus (56) Google Scholar;Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (460) Google Scholar), transcribed from a single gene within EDC on chromosome 1q21, where approximately 25 genes involved in epidermal differentiation are known to reside (Backendorf and Hohl, 1992Backendorf C. Hohl D.A. A common origin for cornified cell envelope proteins?.Nature Genet. 1992; 2: 91Crossref PubMed Scopus (87) Google Scholar;Volz et al., 1993Volz A. Korge B.P. Compton J.G. Ziegler A. Steinert P.M. Mischke D. Physical mapping of a functional cluster of epidermal differentiation genes on chromosome 1q21.Genomics. 1993; 18: 92-99Crossref PubMed Scopus (131) Google Scholar;Gibbs et al., 1993Gibbs S. Fijneman R. Wiegant J. van Kessel A.G. de Putte P. Backendorf C. Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins.Genomics. 1993; 16: 630-637Crossref PubMed Scopus (174) Google Scholar;Mischke et al., 1996Mischke D. Korge B.P. Marenholz E. Volz A. Ziegler A. Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("Epidermal Differentiation Complex") on human chromosome 1q21.J Invest Dermatol. 1996; 106: 989-992Crossref PubMed Scopus (393) Google Scholar;Marenholz et al., 1996Marenholz I. Volz A. Ziegler A. Davies A. Ragoussis I. Korge B.