An Atypical Form of Bullous Congenital IchthyosiformErythroderma is Caused by a Mutation in the L12 LinkerRegion of Keratin 1

Hannie Kremer, Adriana P.M. Lavrijsen,* W.H. Irwin McLean,† E. Birgitte Lane,† Diana Melchers,‡Dirk J. Ruiter,§ Edwin C.M. Mariman, and Peter M. Steijlen‡Department of Human Genetics, University Hospital Nijmegen, Nijmegen, The Netherlands; *Department of Dermatology, Leiden University Medical Center,Leiden, The Netherlands; †CRC Cell Structure Research Group, Department of Anatomy and Physiology, University of Dundee, Dundee, U.K.; Departmentsof ‡Dermatology and §Pathology, University Hospital Nijmegen, Nijmegen, The Netherlands

Defective keratins are the cause of a number of hereditarydisorders of the epidermis and other epithelia. Thedisease-causing mutations in keratins are clustered in therod domain, and mutations in the helix boundary peptidescause the most severe forms of epidermal fragility syn-dromes. Siemens described a family with an atypical, mildform of bullous congenital ichthyosiform erythroderma.Linkage analysis in this family indicated that a defectivetype II keratin might be the underlying cause, keratins

Keratins form the cytoplasmic intermediate filament (IF)network of epithelial cells, including the epidermis andits appendages (Quinlan et al, 1994). The discovery thatdefective keratins are the cause of a number of hereditaryskin disorders has contributed to the elucidation of the

function(s) of the intermediate filaments. In stratified squamous epithe-lia, a major function of the keratins is to impart mechanical integrityto cells.

Epidermolysis bullosa simplex (EBS) is typified by skin blisteringwithin the basal cell layer of the epidermis due to mutations in K5and K14 (Corden and McLean, 1996; Fuchs, 1996). In bullouscongenital ichthyosiform erythroderma (BCIE), the lesions are foundin the suprabasal layers of the epidermis due to mutations in K1 andK10, the predominant keratins in these cell layers. Other keratindisorders of the skin are ichthyosis bullosa of Siemens (K2e), epidermo-lytic palmoplantar keratoderma (K9), focal nonepidermolytic palmo-plantar keratoderma (K1, K16), pachyonychia congenita (K6a, K16,K17), and white sponge nevus (K4, K13) (Corden and McLean, 1996;Fuchs, 1996; Fuchs and Cleveland, 1998).

The disease-causing mutations in the keratins are clustered in specificdomains: H1, the helix initiation peptide in 1A (50% of the mutations),the second part of helix 1A, the L12 linker domain, the C-terminalregion of the helix 2B, and the helix termination peptide. The helixinitiation and termination peptides are the most conserved regions ofkeratins and mutations in these regions cause the most severe formsof the epidermal fragility syndromes (Corden and McLean, 1996;Fuchs, 1996).

Manuscript received February 27, 1998; revised July 16, 1998; accepted forpublication July 20, 1998.

Reprint requests to: Dr. H. Kremer, Department of Human Genetics,University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, TheNetherlands.

Abbreviation: BCIE, bullous congenital ichthyosiform erythroderma.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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K1 and K2e being the best candidates. A substitution ofvaline for aspartic acid was detected at position 340(D340V) in the L12 region of the K1 polypeptide. Themutation was found to cosegregate with the disorder inthe family. Herewith, a genotype-phenotype correlationis shown for bullous congenital ichthyosiform erythro-derma comparable with that described for epidermolysisbullosa simplex. Key words: BCIE/K1. J Invest Dermatol111:1224–1226, 1998

In 1946, during the meeting of the Dutch Society of Dermatologists,a coworker of Siemens presented a family from which he and Siemensthought that the members suffered from ichthyosis bullosa of Siemensbecause of its mild appearence (Botter, 1946). He already recognizedthat this family differed from the families with ichthyosis bullosa ofSiemens by the presence of an erythematous component and thepresence of palmoplantar keratoderma. Later, Siemens changed thediagnosis to BCIE, although he had already concluded that the disordercould be distinguished from the cases described by Brocq and mighteven be a new entity because of the very mild appearance and thepreference for the pressure sites (Siemens, 1970). Here, we have studiedthe descendant of this family and show that this mild phenotype iscaused by a mutation in the linker L12 domain of keratin K1, analogousto mutations in K5 and K14 causing the mild Weber-Cockayne variantof EBS.

MATERIALS AND METHODS

Clinical features of mild BCIE The patients were born with blisters,erosions, and macerated palmar and plantar skin. Except for the erythematousareas due to the erosions, a congenital widespread erythroderma, which iscommon in BCIE, could never be observed. After a few days, scaling wasevident over the whole body and the palmar and plantar skin became morehyperkeratotic. Later in life, the scaling was less severe and diffuse hyperkeratosisof palms and soles was the main complaint. Blistering also decreased and couldoccasionally be provoked by warm humid weather and trauma. Predilectionsites of the mild ichthyosis are the neck, the axillary folds, the shoulders, thehips, and friction sites. The hyperkeratosis often has the appearance oflichenification. In the classification system of DiGiovanna and Bale (1994) thedisorder in this family is best described as of the PS-1 type with small differences.Scaling is generalized in early childhood and localized later in life. Erythrodermais not seen but erythematous areas can be present. Furthermore, blisteringoccurs generalized in early childhood and very rarely, also generalized, in adults(one case).

Histology and electron microscopy Skin biopsies were fixed in 1.5%glutaraldehyde in 1.2 M cacodylate buffer and post-fixed in 1% OsO4 in

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distilled water, dehydrated, and embedded in epoxy. Semi-thin (1 µm) sectionswere used for light microscopy and stained with toluidine blue. Ultrathinsections were prepared for transmission electron microscopy, stained with warmalcoholic uranyl acetate and lead citrate. Haematoxylin and eosin-stained sectionswere used for routine light microscopical examination. Nine biopsies weretaken from three patients, four for electron microscopy and five for routinelight microscopy.

Genetic linkage analysis Polymorphisms within and flanking the keratin Iand II gene clusters were derived from the Genome Data Base. Linkage analysiswas performed as described by Steijlen et al (1994). Statistical analysis wascarried out using the program Linkage (version 5.1) (Lathrop and Lalouel,1984; Lathrop et al, 1984, 1986). Penetrance was assumed to be 100% and thedisease frequency was estimated to be 0.00001.

Mutation detection Mutation analysis of the K2e gene was performed asdescribed (Kremer et al, 1994). For mutation analysis of K1, the followingprimers were used, K1p1 (59-TTC AGG TCT GGG TAC CGA AG-39) andK1p7 (59-CAC CTT GTC AAT GAA GGA GGC AA-39) for the H1 regionand the 1A helix boundary, K1p18 (59-TTC CTT ACA GCA CTC TACC-39) and K1p11 (59-CTT GCT CTG GTA CAA GGA CTC GGC-39) forthe L12 region, and K1p5 (59-TCC ATC AGT GAT GCA GAG CAG CGT-39) and K1p6 (59-CTT ACA CAC ACT CAC GTT CGG GGC-39) for the2B helix boundary region. Cycle sequencing was performed as described inMcLean et al (1994).

Allele-specific oligonucleotide hybridization For allele-specific oligo-nucleotide hybridization, polymerase chain reaction fragments were separatedin 1.5% agarose and blotted onto Hybond N1 (Amersham, Roosendaal, TheNetherlands). Hybridization of the oligonucleotides was in 53SSPE (3 MNaCl, 0.16 M Na2PO4, 0.02 M ethylenediamine tetraacetic acid, pH 7.4), 3%sodium dodecyl sulfate at 55°C. K1LM (59-CTC TCT ATG GTC AAC AACCG-39), K1LW (59-CTC TCT ATG GAC AAC AAC CG-39).

RESULTS

Histology and electron microscopy reveals limited epidermo-lytic hyperkeratosis The histologic and ultrastructural findings arevariable depending on the sampling area. In some ichthyotic lesions,the features of epidermolytic hyperkeratosis can hardly be observed,being limited to the epidermis around the sweat glands (Fig 1a). Otherlesions display mild features of hyperkeratosis, coarse keratohyalinegranules, and mild vacuolization in the upper stratum spinosum;however, the palms, soles, and elbows show marked vacuolization,filament clumping, and acantholysis of the stratum spinosum and thestratum granulosum (Fig 1b). These features were most prominent inthe palms and soles. In total, two of the four biopsies studied withelectron microscopy (Fig 1c) and three of the five biopsies studiedwith light microscopy showed epidermolytic hyperkeratosis.

Linkage analysis indicates a type II keratin defect To investigatewhether this form of BCIE is due to a mutation in a keratin gene, weperformed linkage analysis with markers located within, or close to,the type I (D17S250, KRT10, D17S579) and type II (KRT1, KRT4,Col2A1) keratin gene clusters on chromosome 17q12-q21 and 12q11-q13, respectively. In Table I the pairwise lod scores are given betweenthe markers and the disease.

A strong indication for linkage of the disorder with the type IIkeratin cluster is given by the lod score of 2.53 at θ 5 0.00 with boththe KRT1 and KRT4 marker. The genes of the type I cluster wereexcluded, which is illustrated by the lod scores of –5.01 (θ 5 0.01),–2.58 (θ 5 0.05), and –6.94 (θ 5 0.05) with the markers KRT10,D17S250, and D17S579, respectively. The latter two markers arelocated at no more than 3 cM proximal (D17S250) and distal (D17S579)to the type I cluster (Anderson et al, 1993).

A mutation in the L12 domain of K1 Linkage analysis in thisfamily suggested that the disease is due to a defect in a type II keratin(K1–K8). Both the K1 and the K2e genes are candidates because theyare expressed in the suprabasal layers of the epidermis. The K1 genewas screened for mutations by sequence analysis of the H1 domain,the helix boundary peptides, and the L12 linker region in two patientsand compared with two controls. From the K2e gene only the H1region was analyzed. A heterozygous transversion of adenosine to

Figure 1. Histology and electron microscopy. (a) Photomicrograph of anichthyotic lesion on the hips showing minimal signs of epidermolytichyperkeratosis restricted to the stratum granulosum. Note the coarsekeratohyaline granules around the syringeal opening (arrows). Scale bar: 100 µm.(b) Extensive signs of epidermolytic hyperkeratosis in the whole suprabasalcompartment. Note the coarse keratohyaline granules (arrows) and perinuclearvacuolization (arrowheads). Scale bar: 400 µm. (c) Electron microscopy of skinbiopsy from the sole showing clumping of tonofilaments. Scale bar: 5 µm.

Table I. Two-point lod scores between the disorder and thepolymorphic markers

θ

Marker 0.00 0.01 0.05 0.10 0.20 0.30

COL2A1 1.51 1.48 1.36 1.20 0.86 0.50KRT1/4 2.53 2.49 2.34 2.13 1.64 1.07D17S250 –` –5.88 –2.58 –1.33 –0.36 –0.06KRT10 –` –5.01 –2.30 –1.24 –0.39 –0.09D17S579 –` –13.73 –6.94 –4.20 –1.78 –0.68

1226 KREMER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2. Segregation of the L12 mutation in the family. The mutationis detected by allele-specific oligonucleotide hybridization. The K1LMoligonucleotide exclusively hybridized with DNA of affected individuals.Individuals marked with a small bar are not included in the analysis.

thymidine was detected in the fragment coding for the L12 linkerregion of K1, a nonhelical stretch in the center of the rod domain.This results in a substitution of valine for aspartic acid at position 340of the protein (D340V) and thus in a change of charge.

The segregation of the mutation in the family was studied by allele-specific oligonucleotide hybridization. Oligonucleotides with eitherthe wild-type sequence (K1LW) or the mutated sequence (K1LM)were hybridized to polymerase chain reaction fragments spanning thelinker region, amplified with the primers K1p18 and K1p11. TheK1LW oligonucleotide hybridized to the polymerase chain reactionfragment of all individuals in the pedigree (not shown). The K1LMoligonucleotide sequence hybridized only to the polymerase chainreaction fragments of the affected family members (Fig 2). Additionally,with the latter oligonucleotide, no hybridization could be detected onthe L12 fragments of 50 control persons. This excludes the mutationas a common polymorphism. The mutation is the first detected in theL12 linker region of any keratin other than K5 or K14.

DISCUSSION

Here we describe the first mutation in a patient with BCIE in the L12linker region of K1. So far, mutations causing BCIE were detected inthe H1 region (K1) and the 1A and 2B regions of the rod domain ofeither K1 or K10 (Corden and McLean, 1996; Fuchs, 1996). Thesemutations lead to a more severe phenotype, BCIE of Brocq. Theclinical features of this form of BCIE are markedly milder than thoseof the BCIE of Brocq. A comparable correlation of disease severityand site of mutation has been described for EBS. Mutations in the L12region of K5 and K14 have been described in patients with the milderEBS types of Weber-Cockayne and Koebner, in which no clumpingof keratin filaments is seen in the basal layer of the epidermis (Humphrieset al, 1993; Rugg et al, 1993; Corden and McLean, 1996; Fuchs, 1996).By contrast, mutations in the helix boundary peptides of K5 and K14cause the more severe Dowling Meara form of EBS (Corden andMcLean, 1996; Fuchs, 1996).

Chemical cross-linking studies have led to an alignment model ofkeratin molecules. An overlap is predicted between the helix initiationand termination peptides of two head to tail arranged dimers (Steinertand Parry, 1993; Steinert et al, 1993; Fuchs, 1996). These putativemolecular overlap regions appear to be highly important for filamentelongation. This might explain the high conservation of the rod endsand the severity of the disease in patients with mutations in one ofthese regions. Mutations in the other regions mentioned above, causingmilder phenotypes, may affect lateral associations within the filamentsor interaction with associated proteins (Fuchs, 1996). The disease-causing nature of the mutation indicated by the absence of thesubstitution in 50 nonaffected individuals and its segregation in thefamily is underlined by the conservation of the aspartic acid in all typeII keratins. The substitution of valine for aspartic acid results in achange of charge in the K1 protein that is potentially disruptive. Parryet al have used computer-based molecular modeling and comparativesequence analysis to predict the secondary structure of the L12 linkerdomain, which all intermediate filament proteins possess (North et al,1994). Although the length of the L12 domain varies considerablybetween the members of this protein family, there appears to be a

conserved eight residue sequence motif of alternating polar and apolarresidues, which is highly suggestive of β sheet conformation in a two-chain molecule and/or higher order molecular organization (Northet al, 1994). The D340V substitution reported here replaces a polarwith an apolar residue whithin this conserved eight residue motif.Therefore, the mutation is predicted to be disruptive of the secondary/tertiary structure of the L12 domain, which in turn is likely to bedeleterious to intermediate filament function. We studied structuralimplications of the mutation using different secondary stucture predic-tion algorithms (Geneworks 2.51). No gross changes in structure werecalculated; however, the mutation is predicted to slightly decrease theflexibility of the part of the linker region flanking the mutation. Thishas also been suggested for the V270M mutation in the L12 region ofK14 (Rugg et al, 1993).

In the palms and soles of patients of this family there is markedvacuolization and filament clumping. It is tempting to speculate thatthis is caused by the presence of K9 in palms and soles. The effect ofthe mutation on filament structure might be more deleterious inheterodimers of K1 and K9 than in heterodimers of K1 and K10;however, this may also be due to the higher traumas experienced bypalmoplantar epidermis. So far this mutation is the only representativeof a linker domain mutation in BCIE.

The authors thank the family for their participation in this study and Frank Rietveldfor his technical assistance. This work (W.H.I.M.) was supported by grants from theCancer Research Campaign, The Wellcome Trust, and The Dystrophic EpidermolysisBullosa Research Association (U.K.).

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