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Mutations in Keratin K9 in Kindreds with Epidermolytic Palmoplantar Keratoderma and Epidemiology in Northern Ireland

      Epidermolytic palmoplantar keratoderma (EPPK, MIM #144200) is an autosomal dominant disorder in which hyperkeratosis confined to the palms and soles is characterized histologically by cytolysis of suprabasal keratinocytes. Mutations in the keratin 9 gene (KRT9), a type 1 keratin expressed exclusively in the suprabasal keratinocytes of palmoplantar epidermis, have previously been demonstrated in this disorder. Here, we have studied four Northern Irish kindreds presenting with EPPK. By direct sequencing of polymerase chain reaction products, heterozygous missense mutations in exon 1 ofKRT9 were detected in all the families. These included a novel mutation M156T; as well as M156V in two kindreds; and R162Q in the remaining family. All mutations were confirmed by reverse strand sequencing and restriction enzyme analysis. The point prevalence of EPPK in Northern Ireland was found to be 4.4 per 100,000. To date, all reported EPPK mutations occur in the helix initiation motif at the start of the central coiled-coil rod domain of K9.

      Key words

      Abbreviation:

      EPPK
      epidermolytic palmoplantar keratoderma
      The main function of the intermediate filament network is to resist traumatic damage to the cell (
      • Morley S.M.
      • Lane E.B.
      The keratinocyte cytoskeleton.
      ;
      • McLean W.H.I.
      • Lane E.B.
      Intermediate filaments in disease.
      ). The intermediate filament cytoskeleton of epithelial cells is composed of keratins. Type I and type II keratin proteins form obligate heterodimers through coiled-coil interaction of their rod domains, and these dimers undergo further polymerization to form 10 nm intermediate filaments (
      • Lane E.B.
      Keratins.
      ).
      The keratins are a multigene family that are generally expressed in certain type I/type II keratin pairs in a tissue-specific manner (
      • Lane E.B.
      Keratins.
      ). The type I and type II genes are clustered on chromosomes 12q and 17q. In addition to the main expression pairs, there are a number of accessory keratins expressed in particular epithelial tissues. In suprabasal keratinocytes of the epidermis, keratins K1 and K10 are expressed in all regions of the skin. In palmoplantar epidermis, the accessory keratin K9 is also expressed in suprabasal cells and is presumed to provide additional strength to this specialized epithelial tissue, which undergoes the highest stresses in the human body (
      • Knapp A.C.
      • Franke W.W.
      • Heid H.
      • Hatzfeld M.
      • Jorcano J.L.
      • Moll R.
      Cytokeratin no. 9, an epidermal type I keratin characteristic of a special program of keratinocyte differentiation displaying body site specificity.
      ).
      To date, mutations in a total of 17 keratins have been linked to human diseases (
      • Corden L.D.
      • McLean W.H.I.
      Human keratin diseases: hereditary fragility of specific epithelial tissues.
      ). The most recent additions have been the discovery of K3 and K12 mutations in Meesmann’s corneal dystrophy (
      • Irvine A.D.
      • Corden L.D.
      • Swensson O.
      • et al.
      Mutations in cornea-specific keratins K3 or K12 cause Meesmann’s corneal dystrophy.
      ), the association of a K18 mutation in cryptogenic cirrhosis (
      • Ku N.O.
      • Wright T.L.
      • Terrault N.A.
      • Gish R.
      • Omary M.B.
      Mutation of human keratin 18 in association with cryptogenic cirrhosis.
      ), mutations in hHb6 and hHb1 in monilethrix (
      • Winter H.
      • Rogers M.A.
      • Gebhardt M.
      • et al.
      A new mutation in the type H hair cortex keratin hHb1 involved in the inherited hair disorder monilethrix.
      ,
      • Winter H.
      • Rogers M.A.
      • Langbein L.
      • et al.
      Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix.
      ), and a mutation in K6b in pachyonychia congenita type 2 (
      • Smith F.J.D.
      • Jonkman M.F.
      • van Goor H.
      • Coleman C.
      • Covello S.P.
      • Uitto J.
      • McLean W.H.I.
      Mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2.
      ). Epidermolytic palmoplantar keratoderma (EPPK, MIM #144200) is an autosomal dominant condition characterized by epidermolytic hyperkeratosis confined to the palmoplantar epidermis, first described by Vörner in 1901 (
      • Vörner H.
      Zur Kentniss des Keratoma hereditarium palmare et plantare.
      ). Recently, genetic linkage of EPPK to the type 1 keratin cluster was demonstrated (
      • Reis A.
      • Kuster W.
      • Eckardt R.
      • Sperling K.
      Mapping of a gene for epidermolytic palmoplantar keratoderma to the region of acidic keratin gene cluster at 17q12–q21.
      ) and with the cloning of the K9 cDNA and corresponding gene,KRT9, missense mutations have been identified in a number of EPPK families (
      • Reis A.
      • Hennies H.-C.
      • Langbein L.
      • et al.
      Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).
      ), reviewed recently (
      • Corden L.D.
      • McLean W.H.I.
      Human keratin diseases: hereditary fragility of specific epithelial tissues.
      ). Here, we describeKRT9 mutations in four Northern Irish EPPK kindreds and the epidemiology of the disease in this geographic region.

      Materials and Methods

      Genomic DNA was extracted from whole blood by standard techniques. A 429 bp fragment containing most of exon 1 of theKRT9 gene was amplified using sense primer K9.1L (5′ TTG GCT ACA GCT ACG GCG GAG GAT 3′) and anti-sense primer K9.1R (5′ TGA GAT CAT CAA TAG TGT TAT AAT 3′) in standard polymerase chain reaction (PCR) buffer containing 4% dimethylsulfoxide. The PCR program used consisted of incubation at 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 51°C for 1 min, 72°C for 1 min; and 72°C for 5 min. PCR products were purified using QIAquick PCR purification kit (QIAGEN, Valencia, CA) and were directly sequenced with the amplification primers using the ABI Prism Ready-Reaction system (Perkin-Elmer, Foster City, CA) according to the manufacturer’s recommended protocol. Sequencing ladders were analyzed on an ABI 377 automated sequencer.
      Mutations were confirmed in the four probands by the appropriate restriction digestion ofKRT9 exon 1 PCR products. Nucleotide transition 485G→A (R162Q) creates a recognition site for the restriction enzymePvuII. Both mutations 466A→G (M156V) and 467T→C (M156T) destroy a recognition site for the restriction enzymeNlaIII. The novel mutation M156T (467T→C) was excluded from a population of 50 normal unrelated individuals by restriction analysis. The other mutations have been excluded from control populations in previous studies (
      • Reis A.
      • Hennies H.-C.
      • Langbein L.
      • et al.
      Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).
      ;
      • Hennies H.Q.
      • Zehender D.
      • Kunze J.
      • Kuster W.
      • Reis A.
      Keratin 9 mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Hum.
      ).

      Results

      Diagnostic features of EPPK in the families studied

      Four families with EPPK from Northern Ireland (Figure 1) were diagnosed as having the Vörner form of palmoplantar keratoderma on the basis of clinical appearance and histologic evidence of epidermolytic hyperkeratosis in a number of affected individuals biopsied in the families (not shown). The presence of epidermolytic changes distinguishes EPPK from other palmoplantar keratoderma, such as focal palmoplantar keratoderma, in which pathogenic mutations have been reported in keratin K16 (
      • Shamsher M.K.
      • Navsaria H.A.
      • Stevens H.P.
      • et al.
      Novel mutations in keratin 16 gene underly focal non-epidermolytic palmoplantar keratoderma (NEPPK) in 2 families.
      ). Affected persons in all four families had severe epidermolytic hyperkeratosis of a yellowish appearance, surrounded by a characteristic erythematous border, which was completely confined to the palmoplantar surfaces. The age of onset was in the early months of life.
      Figure thumbnail gr1
      Figure 1Pedigrees of the four EPPK families studied. All kindreds are of Northern Irish origin, a total of 71 living affected persons in this country of population 1.62 million, giving a point prevalence of 4.4 per 100,000.Arrow indicates proband in each case, in whom molecular analysis was carried out.

      Epidemiology of EPPK in Northern Ireland

      Accurate incidence figures for EPPK have not previously been documented in any population. In this study we ascertained all cases of EPPK diagnosed since 1965 in Northern Ireland. In total, 71 living affected patients belonging to four extended kindreds were traced in this population of 1.62 million, giving a point prevalence of 4.4 per 100,000.

      K9 mutations identified in all four kindreds

      Mutation analysis was performed on exon 1 of theKRT9 gene because this exon encodes the helix 1A domain of the K9 polypeptide, where all EPPK mutations have been reported to date (
      • Corden L.D.
      • McLean W.H.I.
      Human keratin diseases: hereditary fragility of specific epithelial tissues.
      ). Direct sequencing ofKRT9 PCR products revealed heterozygous missense mutations in all four families. Only the probands in each kindred (Figure 1) were studied at the molecular level. In family 1, a novel transition mutation 467T→C was detected in the proband, which predicts the amino acid substitution M156T in the K9 polypeptide (Figure 2a). This mutation was confirmed in the proband and was excluded from a population of 50 normal unrelated individuals byNlaIII digestion ofKRT9 PCR products, as shown inFigure 2(b).
      Figure thumbnail gr2
      Figure 2Novel heterozygous missense mutations in exon 1 of the K9 gene in Northern Irish EPPK family 1. (A) In exon 1 of theKRT9 gene, pyrimidine transition mutation 467T→C is seen (arrow), which predicts the amino acid change M156T in the helix 1A domain of the K9 polypeptide (reverse strand sequence shown). (B) Mutation M156T disrupts the recognition sequence for the restriction enymeNlaIII, which was used to confirm the mutation in the proband and exclude it from 50 normal individuals.Lane 1, DNA marker type VI;lane 2,NlaIII digest ofKRT9 exon 1 PCR products derived from a normal person;lane 3,NlaIII digest ofKRT9 exon 1 PCR products derived from the proband in family 1, showing an uncut (upper) band representing the mutant allele.
      In the other three families, direct sequencing revealed that all carry previously reported K9 mutations, which were confirmed by restriction analysis (data not shown). In the probands of both family 2 and family 3, purine transition 466A→G was detected, predicting the amino acid substitution M156V. The proband in family 4 was found to be heterozygous for transition mutation 485G→A, which is predicted to produce amino acid substitution R162Q. All mutations were confirmed both by reverse strand sequencing and by restriction enzyme analysis ofKRT9 PCR products (not shown).

      Discussion

      The Vörner form of palmoplantar keratoderma may be distinguished from other forms by the early onset and diffuse pattern of thick yellowish hyperkeratosis in the absence of associated features (
      • Stevens H.P.
      • Kelsell D.P.
      • Bryant S.P.
      • et al.
      Linkage of an American pedigree with palmoplantar keratoderma and malignancy (palmoplantar ectodermal dysplasia type HI) to 17q24. Literature survey and proposed updated classification of the keratodermas.
      ). The disorder usually affects the hands, which are relatively spared in non-EPPK, and histologic examination reveals epidermolytic changes (
      • Navsaria H.A.
      • Swensson O.
      • Ratnavel R.C.
      • et al.
      Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma.
      ). Here, we diagnosed four Northern Irish families on the basis of these criteria, as part of a epidemiologic study in this province, and have identified K9 mutations in all cases. One of these, M156T, has not previously been reported in K9, although the analogous mutation has been seen in K10 (
      • Paller A.S.
      • Syder A.J.
      • Chan Y.M.
      • Hutton E.
      • Tadini G.
      • Fuchs E.
      Genetic and clinical mosaicism in a type of epidermal nevus.
      ). Because all instances of K9 mutations reported to date occur in the helix initiation motif within the helix 1A domain, mutation detection in EPPK appears to be straightforward (
      • Reis A.
      • Hennies H.-C.
      • Langbein L.
      • et al.
      Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).
      ;
      • Bonifas M.
      • Matsumura K.
      • Chen M.A.
      • et al.
      Mutations of keratin 9 in two families with palmoplantar epidermolytic hyperkeratosis.
      ;
      • Hennies H.Q.
      • Zehender D.
      • Kunze J.
      • Kuster W.
      • Reis A.
      Keratin 9 mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Hum.
      ;
      • Torchard D.
      • Blanchet-Bardon C.
      • Serova O.
      • et al.
      Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer.
      ;
      • Navsaria H.A.
      • Swensson O.
      • Ratnavel R.C.
      • et al.
      Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma.
      ;
      • Rothnagel 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.
      ;
      • Kobayashi S.
      • Tanaka T.
      • Matsuyoshi N.
      • Irnamura S.
      Keratin 9 point mutation in a pedigree of epidermolytic hereditary palmoplantar keratoderma perturbs keratin intermediate filament network formation.
      ;
      • Endo H.
      • Hatamochi A.
      • Shinkai H.A.
      Novel mutation of a leucine residue in coil 1A of keratin 9 in epidermolytic palmoplantar keratoderma.
      ). This part of the keratin polypeptide has been implicated in molecular overlap interactions in keratin polymerization (
      • Steinert P.M.
      • Yang J.M.
      • Bale S.J.
      • Compton J.G.
      Concurrence between the molecular overlap regions in keratin intermediate filaments and the locations of keratin mutations in genodermatoses.
      ), and mutations have been reported in the analogous region of six other type 1 keratins associated with severe epithelial fragility phenotypes. The presence of a CpG-containing arginine codon in this sequence motif, codon 162 in K9, which is conserved in all type I keratins, undoubtedly contributes to the mutational sensitivity of this part of the keratin molecule (
      • Cooper D.N.
      • Krawczak M.
      ). One of these CpG deamination mutations of this arginine, R162Q, was represented in this study; however, in other keratins, mutations associated with severe phenotypes have been found in a second conserved motif at the end of the 2B domain of the protein (
      • Corden L.D.
      • McLean W.H.I.
      Human keratin diseases: hereditary fragility of specific epithelial tissues.
      ), and so exon 6 ofKRT9 is a second potential target for EPPK mutations. Based on other keratin disease studies, it is conceivable that mutations elsewhere in the rod domain might cause a milder form of EPPK, although no mutations of this type are known for K9 at this time.

      Population incidence of EPPK

      By tracing all the EPPK cases in the Northern Irish population, we have been able to calculate a point prevalence of 4.4 per 100,000. The epidemiology of all forms of epidermolysis bullosa simplex has recently been estimated in the Scottish population at 2.86 per 100,000 (
      • Horn H.M.
      • Priestly G.C.
      • Eady Raj
      • Tidman M.J.
      The prevalence of epidermolysis bullosa in Scotland.
      ). This disease is known to be caused by mutations in either K5 or K14 (
      • Corden L.D.
      • McLean W.H.I.
      Human keratin diseases: hereditary fragility of specific epithelial tissues.
      ) and so, based on these figures, the incidence per keratin gene in epidermolysis bullosa simplex is about 1.43 per 100,000, lower than that found here for K9. The reasons for these differences might be due to difficulty in the ascertainment of all cases in the larger Scottish population or skewing of the Northern Irish data due the extensive size of these pedigrees (Figure 1).
      We thank Hans-Jürg Alder and his staff in the Nucleic Acid Facility, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, for DNA sequencing and oligonucleotide synthesis; and Carrie Colernan, Epithelial Genetics Group for technical assistance. This work was supported by the US Public Health Service, National Institutes of Health (grant P01-AR38923), DEBRA UK, and DEBRA of America.

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