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• W2020113731 abstract "The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma. Ultrastructural studies show that intermediate filaments of suprabasal keratinocytes are finer than those of the basal layer. In addition, desmosome numbers are normal, but their inner plaques and midline structures are attenuated. Microsatellite markers were used to screen candidate loci including the epidermal differentiation complex on 1q, the desmoplakin locus on 6p, the type I and II keratin gene clusters on chromosomes 12q and 17q, and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 12q was observed using marker D12S368, with a maximum two-point lod score of 3.496 at a recombination fraction of 0. Direct sequencing of the keratin 1 gene revealed a frameshift mutation in exon 9 that leads to the partial loss of the glycine loop motif in the V2 domain and the gain of a novel 70 amino acid peptide. Using expression studies we show that the V2 domain is essential for normal function of keratin intermediate filaments. The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma. Ultrastructural studies show that intermediate filaments of suprabasal keratinocytes are finer than those of the basal layer. In addition, desmosome numbers are normal, but their inner plaques and midline structures are attenuated. Microsatellite markers were used to screen candidate loci including the epidermal differentiation complex on 1q, the desmoplakin locus on 6p, the type I and II keratin gene clusters on chromosomes 12q and 17q, and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 12q was observed using marker D12S368, with a maximum two-point lod score of 3.496 at a recombination fraction of 0. Direct sequencing of the keratin 1 gene revealed a frameshift mutation in exon 9 that leads to the partial loss of the glycine loop motif in the V2 domain and the gain of a novel 70 amino acid peptide. Using expression studies we show that the V2 domain is essential for normal function of keratin intermediate filaments. striate palmoplantar keratoderma The inherited palmoplantar keratodermas are a clinically varied group of skin disorders that primarily affect palmoplantar epidermis. Striate palmoplantar keratoderma (SPPK; MIM 148700) (Siemens, 1929Siemens H. Keratosis palmo-plantaris striata.Arch Dermatol Syphilol. 1929; 157: 392-408Crossref Scopus (19) Google Scholar;El Sayed and Bazex, 1993El Sayed F. Bazex J. Keratodermie palmoplantaire striee.Ann Dermatol Venereol. 1993; 120: 894-895PubMed Google Scholar) is a rare autosomal dominant skin disorder characterized clinically by linear and focal hyperkeratosis of the palms and soles (Griffiths et al., 1998Griffiths W. Judge M. Leigh I. Disorders of keratinization.in: Rook A. Wilkinson D. Ebling F. Textbook of Dermatology. Blackwell, Oxford1998: 1483-1588Google Scholar;Armstrong et al., 1999Armstrong D.K. McKenna K.E. Purkis P.E. Green K.J. Eady R.A. Leigh I.M. Hughes A.E. Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 143-148Crossref PubMed Scopus (209) Google Scholar), although some phenotypic heterogeneity exists. Genetically, SPPK is heterogeneous as linkage and subsequent mutations have been found at two separate loci. Initially,Hennies et al., 1995Hennies H.C. Kuster W. Mischke D. Reis A. Localization of a locus for the striated form of palmoplantar keratoderma to chromosome 18q near the desmosomal cadherin gene cluster.Hum Mol Genet. 1995; 4: 1015-1020Crossref PubMed Scopus (63) Google Scholar) found linkage to chromosome 18q12 near the desmosomal cadherin gene cluster, comprising the desmoglein and desmocollin genes. This locus has subsequently been supported by the recent detection of several mutations in the desmoglein 1 gene (Rickman et al., 1999Rickman L. Simrak D. Stevens H.P. et al.N-terminal deletion in a desmosomal cadherin causes the autosomal dominant skin disease striate palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 971-976Crossref PubMed Scopus (172) Google Scholar;Hunt et al., 2001Hunt D.M. Rickman L. Whittock N.V. et al.Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.Eur J Hum Genet. 2001; 9: 197-203Crossref PubMed Scopus (84) Google Scholar). The first study to demonstrate a pathogenic mutation in this skin disorder (Armstrong et al., 1999Armstrong D.K. McKenna K.E. Purkis P.E. Green K.J. Eady R.A. Leigh I.M. Hughes A.E. Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 143-148Crossref PubMed Scopus (209) Google Scholar), however, initially showed linkage to the desmosomal plaque protein desmoplakin at chromosome 6p21, and subsequent analysis demonstrated a mutation in desmoplakin (Armstrong et al., 1999Armstrong D.K. McKenna K.E. Purkis P.E. Green K.J. Eady R.A. Leigh I.M. Hughes A.E. Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 143-148Crossref PubMed Scopus (209) Google Scholar). Indeed, this locus has been confirmed by the detection of a second mutation within the desmoplakin gene (Whittock et al., 1999Whittock N.V. Ashton G.H. Dopping-Hepenstal P.J. Gratian M.J. Keane F.M. Eady R.A. McGrath J.A. Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency.J Invest Dermatol. 1999; 113: 940-946Crossref PubMed Scopus (113) Google Scholar). Both desmoglein 1 and desmoplakin are components of specific cell junctions named desmosomes that are involved in cell-cell adhesion and cell communication (Garrod, 1993Garrod D.R. Desmosomes and hemidesmosomes.Curr Opin Cell Biol. 1993; 5: 30-40Crossref PubMed Scopus (254) Google Scholar,Garrod, 1996Garrod D.R. Epithelial development and differentiation: the role of desmosomes.J R Coll Phys Lond. 1996; 30: 366-373PubMed Google Scholar;Kouklis et al., 1994Kouklis P.D. Hutton E. Fuchs E. Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.J Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (239) Google Scholar;Green and Jones, 1996Green K.J. Jones J.C. Desmosomes and hemidesmosomes: structure and function of molecular components.Faseb J. 1996; 10: 871-881Crossref PubMed Scopus (289) Google Scholar) and appear to cause SPPK by haploinsufficiency of their respective proteins. The keratins are a family of structurally related proteins that form intermediate filaments in the cytoplasm of epithelial cells (Quinlan et al., 1994Quinlan R.A. Hutchison C.J. Lane E.B. Intermediate filaments.in: Sheterline P. Protein Profiles. Academic Press, London1994Google Scholar). They are expressed in pairs of type I (acidic), and type II (basic) polypeptides in a tissue- and differentiation-specific manner (Roop, 1995Roop D. Defects in the barrier.Science. 1995; 267: 474-475Crossref PubMed Scopus (166) Google Scholar). Type I/II keratin pairs form heterodimers through association of their α-helical rod domains, and further polymerize to form the characteristic 10 nm filaments (Parry et al., 1977Parry D.A. Crewther W.G. Fraser R.D.B. MacRae T.P. Structure of α-keratin: structural implication of the amino acid sequences of the type I and type II chain segments.J Mol Biol. 1977; 113: 449-454Crossref PubMed Scopus (117) Google Scholar). There are approximately 30 different keratin genes, mutations in 18 of which cause a wide range of diseases affecting skin, nails, hair, and mucosa (Corden and McLean, 1996Corden L.D. McLean W.H. Human keratin diseases: hereditary fragility of specific epithelial tissues.Exp Dermatol. 1996; 5: 297-307Crossref PubMed Scopus (186) Google Scholar;Irvine and McLean, 1999Irvine A.D. McLean W.H. Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation.Br J Dermatol. 1999; 140: 815-828https://doi.org/10.1046/j.1365-2133.1999.02810.xCrossref PubMed Scopus (322) Google Scholar;Ku et al., 2001Ku N.O. Gish R. Wright T.L. Omary M.B. Keratin 8 mutations in patients with cryptogenic liver disease.N Engl J Med. 2001; 344: 1580-1587Crossref PubMed Scopus (135) Google Scholar). Pathogenic mutations in the type I and II epidermal keratins occur within regions thought to be involved in keratin intermediate filament assembly, namely the H1 domain, the helix initiation motif, the L12 linker region, and the helix termination motif (Irvine and McLean, 1999Irvine A.D. McLean W.H. Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation.Br J Dermatol. 1999; 140: 815-828https://doi.org/10.1046/j.1365-2133.1999.02810.xCrossref PubMed Scopus (322) Google Scholar). Mutations in these regions of the suprabasally expressed keratins K1 and K10 underlie bullous congenital ichthyosiform erythroderma/epidermolytic hyperkeratosis (MIM 113800). A missense mutation outside the “hot-spots” has been identified in K1, however, that may affect intermolecular connections rather than filament assembly. In a case of diffuse nonepidermolytic palmoplantar keratoderma a mutation within the V1 domain of K1 (K74I) has been documented (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 (130) Google Scholar). Ultrastructural studies showed a normal tonofilament pattern, although morphologic inspection showed alterations in the tonofilament network organization adjacent to the cornified cell envelope. Further studies demonstrated that this lysine residue is critical for the formation of intermolecular connections between tonofilaments and proteins of desmosomes and the cornified cell envelope via transglutaminases (Kouklis et al., 1994Kouklis P.D. Hutton E. Fuchs E. Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.J Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (239) Google Scholar;Candi et al., 1998Candi E. Tarcsa E. Digiovanna J.J. Compton J.G. Elias P.M. Marekov L.N. Steinert P.M. A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.Proc Natl Acad Sci USA. 1998; 95: 2067-2072Crossref PubMed Scopus (98) Google Scholar). In this study, we investigated a four-generation kindred with SPPK and showed that it is due to a novel frameshift mutation within the V2 domain of K1. We also demonstrate using in vitro transfections that the clinical phenotype in this family is probably a consequence of loss of glycine repeats rather than gain of a novel carboxy terminus. Members of the kindred were examined by the following authors: RM, WAG, RAE, and JAM. Following informed consent, blood samples were available from nine affected and nine unaffected family members. Genomic DNA was extracted from peripheral blood lymphocytes using standard methods. Skin specimens were fixed in half-strength Karnovsky fixative (containing 2% formaldehyde and 2.5% glutar aldehyde in 0.04 M cacodylate buffer), followed by further fixation in 1.3% osmium tetroxide. The samples were then processed with standard transmission electron microscopy techniques and mounted in Epon resin (Tidman and Eady, 1985Tidman M.J. Eady R.A. Evaluation of anchoring fibrils and other components of the dermal-epidermal junction in dystrophic epidermolysis bullosa by a quantitative ultrastructural technique.J Invest Dermatol. 1985; 84: 374-377Crossref PubMed Scopus (188) Google Scholar). For electron microscope examination, ultrathin sections were stained with uranyl acetate and lead citrate and observed in a JEOL 100CX transmission electron microscope. Microsatellite markers were chosen from the Généthon and Marshfield genetic linkage maps (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 (2668) Google Scholar;Broman et al., 1998Broman K.W. Murray J.C. Sheffield V.C. White R.L. Weber J.L. Comprehensive human genetic maps: individual and sex-specific variation in recombination.Am J Hum Genet. 1998; 63: 861-869Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar). Markers D1S305 and D1S498 were used for the epidermal differentiation complex on 1q (Marenholz et al., 1996Marenholz I. Volz A. Ziegler A. Davies A. Ragoussis I. Korge B.P. Mischke D. Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21: chromosomal orientation, new markers, and a 6-Mb YAC contig.Genomics. 1996; 37: 295-302https://doi.org/10.1006/geno.1996.0563Crossref PubMed Scopus (99) Google Scholar), markers DES.MIC1 and DES.MIC3 were used for the desmoplakin gene on 6p (Norgett et al., 2000Norgett E.E. Hatsell S.J. Carvajal-Huerta L. et al.Recessive mutation in desmoplakin disrupts desmoplakin–intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.Hum Mol Genet. 2000; 9: 2761-2766Crossref PubMed Scopus (573) Google Scholar), markers D12S90 and D12S96 were used for the type II keratin cluster on 12q, markers D17S1787 and D17S1868 were used for the type I keratin gene cluster on 17q, and markers D18S463 and D18S536 were used for the desmosomal cadherin gene cluster on 18q. Polymerase chain reactions (PCR) were performed in 7.5 µl containing 100 ng of template DNA, 1 × GeneAmp PCR buffer II (ABI, Foster City), 330 nM of each primer, 250 µM dNTPs, 2.5 mM MgCl2, and 0.30 units of Amplitaq Gold DNA polymerase (ABI) in a Touchdown ThermoCycler (Hybaid, Ashford, U.K.). One of the two primers was 5′ labeled with fluorescent FAM, HEX, or NED dyes (ABI). Microsatellite DNA marker analysis was performed on an ABI377 automated DNA sequencer running Genescan and Genotyper software (ABI). Two-point lod scores were computed using Cyrillic version 2.1.3 (Cherwell Scientific Publishers, Oxford, U.K.) running the MLINK algorithm of LINKAGE version 5.1. The mutant allele frequency was assumed to be 0.001 with 100% penetrance. Allele frequencies were assumed to be equal in the population. Total RNA was isolated from skin biopsy or cultured keratinocytes using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159https://doi.org/10.1006/abio.1987.9999Crossref PubMed Scopus (0) Google Scholar) and subjected to random hexamer primed cDNA synthesis. Exons 1–9 of the human K1 gene were directly amplified from genomic DNA as previously described (Whittock et al., 2000Whittock N.V. Eady R.A. McGrath J.A. Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5.Biochem Biophys Res Commun. 2000; 274: 149-152Crossref PubMed Scopus (19) Google Scholar), except that the reverse primer for exon 9 was replaced with primer K1 e9R3′ 5′-GCA AGG AGC TGT CCA CAC-3′ (antisense). For PCR, 200 ng of patient or control genomic DNA was added to a premix containing PCR buffer [67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4, 1.5 mM MgCl2, 0.17 mg per ml bovine serum albumin (Sigma, Poole, U.K.), and 10 mM 2-mercaptoethanol], 10 nmol of each dNTP, and 20 pmol of each primer in a total volume of 50 µl. After an initial denaturation at 95°C for 2 min, 2.5 units of Taq polymerase (Promega, Madison, WI) were added followed by 35 cycles of 95°C for 10 s, annealing temperature for 10 s, 72°C for 30 s, with a final incubation of 72°C for 5 min. The PCR products were examined by 1% agarose gel electrophoresis, purified using spin columns (Qiagen, Crawley, U.K.), and directly sequenced using Big Dye terminators on an ABI 3100 genetic analyzer (ABI). To study the inheritance pattern of the mutation within the family and unrelated normal controls, fluorescent PCR was performed using primers K1 e9F (Whittock et al., 2000Whittock N.V. Eady R.A. McGrath J.A. Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5.Biochem Biophys Res Commun. 2000; 274: 149-152Crossref PubMed Scopus (19) Google Scholar) (sense) and K1 e9R5′ 5′-FAM-CCT CCA GAG CCA TAG CTG-3′ (antisense) to amplify a 251 bp fragment. Fragment analysis was performed on an ABI 377 automated DNA sequencer running Genescan and Genotyper software as described above. To determine whether the mutant allele was expressed at the transcript level, mRNA was isolated from cultured cells and single stranded cDNA was synthesized as above. Primers K1 e8/9 5′-GTG AGT GTG TCT GTG AGC-3′ (sense) and K1 e9R5′ 5′-CCT CCA GAG CCA TAG CTG-3′ (antisense) were used to amplify a 209 bp fragment using the above conditions. The PCR products were examined by 1% agarose gel electrophoresis, purified using spin columns, and directly sequenced as above. To determine whether the clinical phenotype was due to the loss of the V2 domain or the gain of a novel peptide, we performed site-directed mutagenesis on a K1-GFP cDNA construct (gift of Sarah Hatsell and David Kelsell, Center for Cutaneous Research, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, London) using the QuikChange Kit (Stratagene) according to the manufacturer's instructions. The K1-GFP construct was made by inserting the K1 cDNA between the EcoR1 and Pst1 sites of the vector pEGFP-C3 (Clontech, Palo Alto, CA). Primers used for the 1628delG mutation described in this study were 5′-GCT ATG GTT CTG GAG TGG CGG CGG C-3′ (sense), and 5′-GCC GCC GCC ACT CCA GAA CCA TAG C-3′ (antisense). Primers used to insert a stop codon at the start of the second glycine repeat (1624 G→T) were 5′-GCT ATG GTT CTT GAG GTG GCG GCG GC-3′ (sense), and 5′-GCC GCC GCC ACC TCA AGA ACC ATA GC-3′ (antisense). Primers used to insert a stop codon at the end of the H2 domain (1540 G→T) were 5′-CAC CAC CAT CAG TTG AGG TGG CAG CC-3′ (sense), and 5′-GGC TGC CAC CTC AAC TGA TGG TGG TG-3′ (antisense). Primers used to replicate the V2 domain mutation described by Sprecher (Sprecher et al., 2001Sprecher E. Ishida-Yamamoto A. Becker O. et al.Evidence for novel functions of the keratin tail emerging from a mutation causing ichthyosis hystrix.J Invest Dermatol. 2001; 116: 511-519https://doi.org/10.1046/j.1523-1747.2001.01292.xCrossref PubMed Scopus (95) Google Scholar) (1609 GG→A) were 5′-GGC TCC GGA GGT ATA GCT ATG GTT CTG G-3′ (sense), and 5′-CCA GAA CCA TAG CTA TAC CTC CGG AGC C-3′ (antisense). HaCaT cells (spontaneous human keratinocyte cell line) were grown on 13 mm round coverslips in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 100 U per ml penicillin, and 100 µg per ml streptomycin until approximately 50% confluent. The cells were transfected with K1-GFP constructs using Fugene 6 (Roche Molecular Biochemicals, Lewes, U.K.) according to the manufacturer's instructions. Cells were fixed 24 h after transfection in 3% paraformaldehyde and permeabilized with 0.2% Triton X-100. Cells were stained with 4,6-diamidino-2-phenylindole (1 µg per ml; Sigma) for 5 min at room temperature and mounted in hydromount containing 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma). Cells were visualized using an Olympus BX50 microscope with MacProbe v 4.0.1 software (Perceptive Scientific Instruments, League City, TX). A single four-generation family of British origin with 14 individuals affected with PPK was studied (Figure 1). The inheritance pattern of PPK in the family was consistent with an autosomal dominant mode of inheritance, exhibiting male-to-male trans mission. Affected individuals presented during early childhood with a phenotype that exhibits SPPK on the palms and more diffuse changes on the soles (Figure 2). There was no involvement of nonpalmoplantar skin, however, and both hair and nails were normal.Figure 2Clinical appearance of the SPPK family. (a) The affected proband and his two sons show focal keratoderma on the palms and some linear hyperkeratosis along the fingers. (b) The proband shows focal hyperkeratosis on the soles.View Large Image Figure ViewerDownload (PPT) Ultrastructural studies on lesional skin showed that the stratum corneum and nucleated epidermal layers were thickened. Keratin intermediate filaments within basal keratinocytes appeared normal, in contrast to those of the spinous layers, which appeared to be much less electron dense and possibly shorter than normal, although the cell shape and size appeared normal (Figure 3). Ultrastructural studies on nonlesional skin also demonstrated less electron dense keratin filaments in the spinous layer (not shown). Desmosomes appeared to be present in normal numbers, but their outer plaques (where the tonofilaments insert) and midline structures appeared attenuated. DNA was prepared from 18 family members, including nine affected individuals, and tested for genetic linkage using a panel of microsatellite markers located near candidate genes involved in the structure and/or development of epithelial tissues. Markers located on chromosomes 1q, 6p, 17q, and 18q were excluded for linkage to the disease, thereby indicating that the gene for SPPK in this study was not located in the epidermal differentiation complex, the desmoplakin locus, the type I keratin gene cluster, or the desmosomal cadherin gene cluster, respectively (data not shown). In contrast, significant linkage was seen with a number of markers located in the vicinity of the type II keratin gene cluster on chromosome 12q, as shown in Table I. Maximum two-point lod scores of 3.496 and 3.311 at θ = 0 were obtained for the markers D12S368 and D12S83, respectively (Table I). Visible recombination events in the family excluded the PPK gene from the region centromeric to D12S85 and telomeric to D12S326. Hence, the genetic defect in this family is located between the microsatellite markers D12S85 and D12S326, which encompass the type II keratin gene cluster (http://www.genome.ucsc.edu).Table ISPPK family: two-point lod scores with 12q markersaSex-averaged genetic distances in centiMorgans (cM) from the Marshfield Genetic Map.ZmaxMarkerDistanceθ = 00.050.100.150.200.250.300.350.400.450.50D12S34553.09–∞1.9191.9381.8171.6271.3901.1170.8180.5090.2210.000D12S166356.38–∞1.7431.7631.6431.4571.2280.9710.6990.4320.1940.000D12S8561.34–∞1.7441.7871.6901.5261.3161.0700.7970.5090.2320.000D12S170162.541.1781.0900.9980.9020.8010.6930.5770.4510.3150.1650.000D12S36866.033.4963.2082.9052.5842.2451.8851.5051.1060.7010.3180.000D12S9668.161.4541.4031.3101.1871.0390.8710.6850.4870.2900.1160.000D12S9071.611.7801.5811.3731.1580.9360.7100.4890.2910.1410.0490.000D12S8375.173.3113.0442.7622.4642.1491.8141.4591.0860.7030.3310.000D12S31379.932.7582.4932.2141.9211.6131.2910.9600.6340.3400.1190.000D12S32686.40–∞0.2690.4360.4710.4480.3900.3090.2160.1220.0440.000D12S35195.56–∞1.0801.1751.1341.0310.8860.7120.5170.3140.1310.000a Sex-averaged genetic distances in centiMorgans (cM) from the Marshfield Genetic Map. Open table in a new tab Of the type II keratin genes, those expressed in palmoplantar skin were considered as candidates. Due to the fact that the keratin intermediate filaments of the spinous layer appeared abnormal, however, we decided to concentrate initially on the K1 gene KRT1. Genomic DNA from one affected and one unrelated individual was used to PCR amplify each of the nine exons of KRT1 using conditions previously described, followed by direct sequencing. Sequence analysis of exons 1–8 revealed no potential pathogenic mutations; however, analysis of exon 9 revealed a deletion of a G nucleotide at position 1628 (1628delG with reference to GenBank accession no. NM_006121) (Figure 4). Specific fluorescent PCR demonstrated that the deletion segregated with the disease in the family studied, but was not present in 100 unrelated ethnically matched control chromosomes. In addition, the fluorescent PCR encompassed the 21 bp deletion polymorphism that results in the deletion of a complete glycine loop motif as described byKorge et al., 1992aKorge B.P. Compton J.G. Steinert P.M. Mischke D. The two size alleles of human keratin 1 are due to a deletion in the glycine-rich carboxyl-terminal V2 subdomain.J Invest Dermatol. 1992; 99: 697-702Abstract Full Text PDF PubMed Google Scholar). Analysis demonstrated that although some affected family members had also inherited the 21 bp deletion polymorphism it was not on the same allele as the 1628delG mutation and therefore did not cosegregate with the disease-causing mutation. Screening of unaffected normal controls for the 21 bp polymorphism yielded a polymorphism information content (PIC) of 0.39, thus indicating its usefulness for linkage analysis. The mutation 1628delG is predicted to cause a frameshift with a subsequent premature stop codon to be inserted 70 amino acids downstream of the deletion (Figure 5). This region of K1 has been postulated to anchor intermediate filaments to the cornified cell envelope. To determine whether the mutant transcript had been degraded we used reverse transcription PCR (RT-PCR) on mRNA extracted from patient cultured keratinocytes. Direct sequencing of the RT-PCR product demonstrated both mutant and wild-type alleles (Figure 4), therefore indicating that although the mutant allele contained a premature termination codon it had not been significantly degraded by nonsense mediated mRNA decay. To determine whether the pathology of the disease is due to the loss of glycine loops of K1 or the gain of a novel peptide, several K1-GFP constructs were made using site-directed mutagenesis. The mutation 1628delG described in this study, as well as 1609 GG→A recently characterized in a case described as ichthyosis hystrix Curth-Macklin (Sprecher et al., 2001Sprecher E. Ishida-Yamamoto A. Becker O. et al.Evidence for novel functions of the keratin tail emerging from a mutation causing ichthyosis hystrix.J Invest Dermatol. 2001; 116: 511-519https://doi.org/10.1046/j.1523-1747.2001.01292.xCrossref PubMed Scopus (95) Google Scholar), 1540 G→T to create a stop codon after the H2 rod domain, and 1624 G→T to create a stop codon within the fourth glycine loop motif were constructed (Figure 6). These constructs were transfected into HaCaT cells that already possess suprabasal K1/10 intermediate filaments to determine whether their expression disrupted filament formation or localization. GFP vector control alone demonstrated a widespread diffuse distribution (not shown). The wild-type K1 showed a normal filamentous distribution forming desmosomal contacts between cells (Figure 7a). Although the four mutant constructs showed some cytoplasmic filament staining that appeared less dense, however, most of the staining was in a nuclear localization in each case (Figure 7b–e). It could therefore be concluded that the nuclear localization observed is due to the loss of the wild-type V2 and not the gain of the novel peptide seen in the two frameshift mutants (1609 GG→A and 1628delG).Figure 7K1-GFP expression studies. Wild-type staining is filamentous (a), whereas the staining is largely nuclear for the mutants 1628delG (b), 1609GG→A (c), 1540G→T (d), and 1624 G→T (e). Scale bar: 7 μm.View Large Image Figure ViewerDownload (PPT) Here we report a kindred affected with SPPK that is caused by a frameshift mutation within the V2 domain of K1, resulting in a novel apolar α-helical forming peptide. The V2 domains of the terminally differentiating epidermal keratins K1 and K10 are unusually glycine rich in comparison with other keratins. These glycine-rich sequences follow the form X(Y)n where X is usually an aromatic or aliphatic residue, Y is usually glycine and n is highly variable between 1 and 35 residues. These glycine-rich sequences are predicted to form loops by the grouping of intervening aliphatic amino acids (Steinert et al., 1991Steinert P.M. Mack J.W. Korge B.P. Gan S.Q. Haynes S.R. Steven A.C. Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins.Int J Biol Macromol. 1991; 13: 130-139Crossref PubMed Scopus (156) Google Scholar). Previous studies have shown that glycine loops are highly insoluble and their presence may confer flexibility within the cell (Steinert, 1993Steinert P.M. Structure, function, and dynamics of keratin intermediate filaments.J Invest Dermatol. 1993; 100: 729-734Abstract Full Text PDF PubMed Google Scholar). In basal cells there are reduced numbers of loops in K5 and K14 indicating that flexibility within the basal cells may be tolerated. The glycine loop motif is also very prominent in loricrin, a protein that constitutes approximately 70% of the cornified cell envelope of terminally differentiated epidermis (Hohl et al., 19" @default.
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