A Premature Stop Codon Mutation in the 2B Helix Termination Peptide of 5 in a German Epidermolysis Bullosa Simplex Dowling–Meara Case

Felix B. Mu¨ller, Ingrun Anton-Lamprecht,* Wolfgang Ku¨ster,† and Bernhard P. Korge Department of Dermatology and Venereology, University of Cologne, Germany; *Institute for Ultrastructure Research of the Skin, University of Heidelberg, Germany; †TOMESA-Clinic for Allergy, Skin and Joint Diseases, and Rheumatism, Bad Salzschlirf, Germany

Epidermolysis bullosa simplex (EBS) is caused by of the 2B helix) in the last residue of the highly defective assembly of keratin intermediate filaments conserved helix-termination peptide K/LLEGE of the in basal keratinocytes and recent studies indicated 2B rod domain of keratin K5. This represents the first causal mutations in the keratin KRT5 and KRT14 premature stop codon mutation identified within the genes. In this study, we describe a novel KRT5 muta- K/LLEGE motif of any disorder reported so far that tion in a German sporadic case of EBS Dowling– is caused by keratin mutations. Key words: epidermolysis Meara. Transition of G to T (nucleotide position 2334) bullosa simplex/intermediate filaments/keratin genes. J Invest leads to a premature stop codon (E477stop, residue 93 Dermatol 112:988–990, 1999

pidermolysis bullosa simplex (EBS) is a group of heredit- in the 1A helix initiation peptide of K14 or in the 2B helix ary skin disorders with mostly autosomal dominant termination peptide of K5. Here we report a novel K5 mutation inheritance, characterized by intraepidermal blister (E477stop, residue 93 of the 2B helix, the last residue of the K/ formation due to cytolysis of basal keratinocytes after LLEGE motif) in a patient showing the characteristic clinical and minor mechanical stress. Blistering is more frequent in ultrastructural features of EBS-DM. The location of this K5 warmE and humid weather and generally improves with advancing mutation is consistent with previous reports but also provides age. Epidermolysis bullosa herpetiformis Dowling–Meara (EBS- additional in vivo evidence that partial K/LLEGE truncation of K5 DM) is the most severe subtype of EBS, characterized by extensive indeed disturbs normal K5/K14 keratin filament assembly, which blistering since birth with herpetiform marginal spreading and is in agreement with the in vitro assembly studies by Wilson central healing, leaving light-brown postbullous pigmentations. Oral et al (1992). mucosal involvement, nail dystrophy, onychogryposis, formation of milia, and palmoplantar are common features MATERIALS AND METHODS (Anton-Lamprecht and Schnyder, 1982; Fine et al, 1991; Gedde- Case report (Fig 1) A 38 y old woman was affected since birth by Dahl and Anton-Lamprecht, 1996). In contrast to the milder forms generalized blister formation with herpetiform spreading and central healing, of EBS, skin biopsies of EBS-DM reveal distinctive ultrastructural leaving faint brown postbullous pigmentations. Hemorrhagic blisters abnormalities: aggregation and clumping of basal resulting occurred on trunk and extremities including palms and soles, on the face, in a total collapse of the keratin of basal keratinocytes and on the oral mucosa after minor trauma. Some improvement was noted (Anton-Lamprecht and Schnyder, 1982; Anton-Lamprecht, 1983, after the fourth year of life. Palmar and plantar hyperkeratoses were 1992). reported to have existed already before walking and often caused painful In 1991, Vassar et al (1991) showed that expression of a truncated problems in periods of plantar blistering. Subungual blisters and irregular nail plate thickening were present, and the patient experienced recurrent K14 gene in transgenic mice led to clinical and morphologic generalized blistering even in adulthood. The diagnosis of the Dowling– findings resembling EBS-DM. Subsequent sequence analysis of Meara type was stated by electron microscopy of a skin biopsy sample at keratin genes in EBS patients revealed that point mutations in the the age of 19 and confirmed clinically some time later on clinical inspection basal keratins K5 and K14 are clustered in distinct regions of the in Heidelberg. As no other family member is affected, she represents a central rod domain (for review see Corden and McLean, 1996; spontaneous new DM mutation. Her son is unaffected. Fuchs, 1996; Korge and Krieg, 1996; Paller, 1996). Almost all EBS-DM mutations are located in the highly conserved hydrophilic Electron microscopy A 1 d old blister induced experimentally by peptides of either the amino or the carboxy end of the central α- friction by the patient, had been excised elsewhere for electron microscopy helical rod domain with the majority of mutations residing either at the age of 19 y. The sample was subdivided prior to fixation, then fixed in phosphate-buffered 2.5% glutaraldehyde solution, dehydrated in graded ethanols, and embedded in Durcupan. Cured tissue blocks were later sent Manuscript received October 9, 1998; revised January 15, 1999; accepted to Heidelberg for electron microscopic analysis and diagnosis. Ultrathin for publication March 7, 1999. sections were investigated using a Philips EM 400 electron microscope. Reprint requests to: Dr. Bernhard Korge, Klinik und Poliklinik fu¨r Dermatologie und Venereologie, Universita¨tzuKo¨ln, 50924 Ko¨ln, Mutation detection Genomic DNA isolated from ethylenediamine Germany. tetraacetic acid blood samples was used to amplify exon 7 of KRT5 by Abbreviations: KRT5, gene; KRT14, gene; EBS- polymerase chain reaction (Taq DNA polymerase, Boehringer, Mannheim, DM, epidermolysis bullosa simplex Dowling–Meara. Germany). The primer set was 5Ј CCC ACT CTC CTC CTT TCT

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Figure 1. Pedigree of the EBS-DM case. Family members analyzed are indicated by numbers. Figure 2. Electron microscopy. Part of a basal cell with clumping of ATC 3Ј and 5Ј CGC TTT ATC AAC TGA AGG CC 3Ј.A50µl reaction the keratin network: keratin clumps (asterisks) are rounded or irregular in solution containing 250 ng DNA, 200 nM of each primer, 200 µMof shape and only partly connected to desmosomes; the remainder of the each dNTP, 10 mM Tris–HCl, 1.25 U Taq DNA polymerase with cytoplasm with nucleus (N), mitochondria (M), ER cisternae, and melanin optimized pH, MgCl2 and KCl concentrations were initially denaturated granules is ultrastructurally normal, although devoid of a normal keratin at 94°C for 2 min, followed by 35 cycles of 1 min at 94°C, 1 min at 56°C cytoskeleton. Final magnification, ϫ17600; scale bar:1µm. and 1.5 min at 72°C, followed by a 7 min final extension step at 72°C. Direct sequencing of polymerase chain reaction products was preceded by shrimp alkaline phosphatase and exonuclease 1 treatment (Sequenase PCR product sequencing kit, USB/Amersham Life Science, Cleveland, OH) as described (Mu¨ller et al, 1998). An additional sequencing primer was 5Ј CAA GCA GGA CAT GGC CCG 3Ј. RESULTS Skin ultrastructure Advanced blister formation with partial or complete destruction of basal cells, small intrabasal blisters, and exocytosis of lymphocytes were seen in the blister area. Most affected basal cells were necrotic. In the upper dermis, increased numbers of small capillaries, a lymphohistiocytic infiltrate, and pigment-loaded melanophages reflected the intense inflammatory reaction to blister formation. In less destroyed areas and adjacent to the blister basal cells revealed round or irregular clumps and aggregation of their keratin filaments as a result of a collapse of the entire basal keratin network. Most of the clumps were distributed irregularly in the cytoplasm that otherwise was entirely normal, and only few of them were attached to desmosomes (Fig 2). Upper epidermal keratinocytes were normal apart from some unspecific high-level paranuclear vacuoles due to friction. Clumping of basal keratins is unique among EBS subtypes and pathognomonic of the DM type, thus permitting the diagnosis of EBS-DM for the patient. Mutation analysis The entire H1, 1A, and 2B domains of keratin K5 and K14 were screened for potential mutations (Fig 3). The sequence analysis detected a G to T transition at base 2334 Figure 3. Mutation E477stop. Part of the DNA sequencing gel showing (EMBL accession code: M28496) in residue 93 of the 2B helix of the point mutation detected in the helix termination peptide of K5. KRT5 leading to an exchange of a glutamic acid to a stop codon Sequences of the patient (A) and an unaffected family member (U) are at position 477 of K5 (2B rod domain position 93, the last residue shown. Arrow indicates the location of the mutation. of the K/LLEGE motif). This premature stop codon results in a truncated K5 protein lacking the last four amino acid residues of mutations the truncated K14 proteins as well as the corresponding the helix termination peptide (residues 477–480) and the entire tail mRNA are obviously unstable, which results in a functional K14 domain. The parents, the two sisters, and the son of the patient, knockout in homozygous individuals. In heterozygous individuals who were all clinically unaffected, did not show the mutation and the defects can be compensated by the remaining unaffected K14 neither did 23 normal nonrelated controls. gene. In contrast, the stop codon mutation in K5 found in our autosomal dominant case most probably leads to a stable truncated DISCUSSION protein which participated in intermediate filament formation. This EBS-DM mutations are generally located in the highly conserved notion is supported by our electron microscopic data, revealing in hydrophilic residues of either end of the central α-helix, mainly in our patient the basal clumping of keratins which is characteristic the helix termination peptide of K5 or in the helix initiation for EBS-DM, while basal keratins were missing in both cases of peptide of K14. Previously, two stop codon mutations were also K14. How the defective keratins impair normal keratin intermediate reported in the 1A/1B region of K14 in two patients with autosomal filament (KIF) stability is still speculative. Several lines of experi- recessive EBS (Chan et al, 1994; Rugg et al, 1994). In these mental evidence suggest that the mutation described here will alter ¨ 990 MULLER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY various stages of KIF formation and help to understand the rather It will be of interest for the future whether corresponding severe clinical phenotype of our patient. mutations can be found in other disorders caused by keratin defects. The basic feature of intermediate filaments including keratins is the periodicity of heptad repeats in the central α-helix, which allows to form a coiled-coil dimer (Cohen and Parry, 1986). The We are grateful to the family for their willing co-operation. Supported by the ‘Verein residues in the a and d position are mostly occupied by apolar der Freunde und Fo¨rderer der Universita¨tzuKo¨ln e.V.’ (BPK) and the ‘Ko¨ln amino acids which form a hydrophobic core between interacting Fortune Program/Faculty of Medicine/University of Cologne’ (BPK). coiled-coil molecules. Oppositely charged residues in the e and g position are able to stabilize the coiled-coil by forming salt bridges (Conway and Parry, 1990). Site-directed mutagenesis of the d REFERENCES position at the end of the 2B rod domain of K8/K18 caused the Anton-Lamprecht I: Genetically induced abnormalities of epidermal differentiation heptad structure to dissolve and perturbed heterodimer formation and ultrastructure in ichthyoses and epidermolyses; pathogenesis, heterogeneity, in vitro (Hatzfeld and Weber, 1990). Based on these observations fetal manifestation, and prenatal diagnosis. J Invest Dermatol 81:149s–156s, 1983 the occurrence of a premature stop codon mutation in the d Anton-Lamprecht I. The skin. In: Papadimitriou JM, Henderson DW, Spagnolo DV position of the K5 helix termination peptide as reported here (eds). Diagnostic Ultrastructure of Non-neoplastic Diseases. 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