REPORTS Ichthyosis Bullosa of Siemens-A Disease Involving Keratin 2e

W.H. Irwin McLean, Susan M. Morley, E. Birgitte Lane, Robin A.J. Eady, * W. Andrew D. Griffiths, * David G. Paige,t John 1. Harper,t Caroline Higgins,:j: and Irene M. Leigh:j: CRe Cell Structure Research Group, Cancer Research Campaign Laboratories, Department of Anatomy & Physiology, Medical Sciences Institute, University of Dundee, Dundee; ·St. John's Institute of Dermatology, United Medical and Dental Schools, St.Thomas's Hospital; tDermatology Department, The Hospital for Sick Children; and :I:Experimental Dermatology Laboratories, Medical College of the Royal London Hospital, London, U.K.

Ichthyosis bullosa of SieInens (IBS) is a congenital Inutation in the highly conserved helix terInina­ bullous ichthyosis without erythroderIna. In contrast tion Inotif, producing the protein sequence change to bullous congenital ichthyosiforIn erythroderIna LLEGEE-LLEGKE. This Inutation was found in all (BeIE), there is a relatively Inild involveInent of the affected IneInbers of a five-generation kindred and skin and epiderInolytic hyperkeratosis (EHK) is re­ also in a sporadic case in a second unrelated faInily. No stricted to the upper suprabasallayers ofthe epiderInis. Inutation was seen in unaffected individuals. The InU­ TonofilaInent aggregation was observed by EM in su­ tation destroys a Mnll restriction site, which allowed prabasal cells froIn affected patients in the two faIni­ exclusion of the Inutation froIn a population of 50 un­ lies under study, indicative of a keratin abnorInality. affected unrelated individuals by restriction fragInent Keratin 2e is a differentiation specific type II keratin analysis ofK2e peR products. This is the sixth keratin expressed suprabasally in the epiderInis. Part of the gene found to be involved in an inherited epiderInal K2e gene was aInplified by polYInerase chain reaction disorder. Keywords: epiJermolytic hyperkeratosis (EHK) / using genoInic DNA froIn affected and unaffected in­ intermediate filaments/keratin mutation /K2 / epidermis/ dif­ dividuals froIn two IBS faInilies. Direct sequencing of ferentiation.] Invest Dermatol103:277-281, 1994 polymerase chain reaction products revealed a point

chthyosis bullosa of Siemens (IBS) (MIM number 146800 pressed specifically in hard palate (9). Recently, the K2e eDNA [1]) is a mild type of epidermolytic ichthyosis first described sequence was determined [8). K2e is most similar to Kl in structure, by Siemens in 1937 (2) as a separate disorder from bullous consistent with its suprabasal expression pattern, which is reported congenital ichthyosiform erythroderma of Brocq (BClli, to appear in the third or fourth cell layers of the epidermis, continu­ MIM number 113800 [1 D. The phenotype of this disorder ing outwards. It is expressed in epidermis from a number of body wasI further delineated more recently [3 - 5) . The disease is charac­ sites [8). terised by mild changes particularly on flexures, shins, and perium­ The role of keratins in maintaining the structural integrity of the bilical region. Blistering occurs in response to mild physical trauma epidermis has come to light in recent years with the finding that and results in superficial erosion or "moulting" of the outer skin. point mutations in K5 and K141ead to various forms ofepidermoly­ Erythroderma is not present. Keratin filament aggregates are seen sis bullosa simplex (EBS). In EBS, cytolysis occurs in the basal cell by electron microscopy in the granular and upper spinous layers of layer where KS and K14 are specifically expressed. Similarly, muta­ the epidermis. tions in K1 or Kl0 lead to suprabasal cytolysis and results in bullous The cytoskeleton of epithelial cells is largely composed of keratin congenital ichthyosiform erythroderma (BClli), also called epider­ intermediate filaments. In epidermis, the basal cells express K5 (type moly tic hyperkeratosis (EHK) (recently reviewed (10)). More re­ II) and K14 (type I) predominantly. In suprabasal cells, synthesis of cently, point mutations in the palm and sole specific K9 have been these proteins is replaced by differentiation-specific keratins Kl shown to be responsible for epidermolytic palmoplantar kerato­ (rype II) and Kl0 (type I) (6) . An additional type II keratin, K2e, is derma (EPPK) (11). Here we describe a mutation in K2e associated expressed in epidermis [7,8). This is a protein of molecular weight with IBS in two unrelated British families. 65.8 kDa encoded by a 2.6-kb mRNA species. Another keratin of MATERIALS AND METHODS similar molecular weight (K2p) has been shown to be of a different amino acid composition, is encoded by a different gene, and is ex- Light and Electron Microscopy Biopsy sa mples of clinically affected skin from four members of family 1 and the si ngle affected member of family 2 were processed for light and electron microscopy as described previously Manuscript received April 21, 1994; revised May 25, 1994; accepted for [12). Briefly, the samples were fixed first in half-strength Karnovsky fixa­ publication June 10, 1994. tive, then in 1.3% aqueous osnuum tetroxide and then dehydrated in ethanol Reprint requests to: Dr. W .H.1. McLean, CRC Laboratories, Department and embedded in epoxy resin (Taab L1boratories Equipment Ltd., Alder­ of Anatomy and Physiology, Medical Sciences Institute, University of Dun­ maston, Berkshire, UK). Semithin (1 f.lm) and ultrathin (- 80 nm) sections dee, Dundee 001 4HN, U.K. were cut on a Reichert OMU4 ultramicrotome (Leica UK Ltd., Milton Abbreviations: BCIE, bullous congenital ichthyosiform erythroderma; Keynes, UK). Semithin sections were stained with methylene blue and azure lBS, ichthyosis bullosa of Siemens. II (Richardson) for li ght microscopy. Ultrathin sections were stained with

0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc.

277 278 McLEAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY lead citrate and uranyl acetate and viewed in a lEOL 100 CX transmission IBS Family 1 lBS Family 2 electron microscope, operating at 80 kV. Ultrathin sections of normal epi­ dermis were prepared and examined for comparative purposes. Polymerase Chain Reaction (PCR) A l.S-kb fragment (approxi­ mately) of the K.2e gene was amplified from genomic DNA using primers [J II rt; K2-S (5' GCA GTG T AA GAA TGT GCA AGA TG 3', + strand) and K2-6 111 °, 11 2 (5' CAG TCA CAT TGC TGC TGA GG 3', - strand). Primers were designed from the cDNA sequence [8) using the program PRIMER version 0.5 (1 3) and, due to the high degree of conservation between K.2e and Kl, III primers were checked aga inst a K 1 /K2e DN A alignment for K2e specificiry. Due to the proximiry of the helix termination sequence to the predicted position of intron VII, this intron is included in the K.2-S/K2-6 fragment. limon VII is about 1.25 kb in size. PCR was performed in 100-/11 reaction IV volumes with standard buffer with 1 mM MgCI2 and 10% dimethylsulfox­ ide and 1 U of Amplitaq polymerase (Perkin-Elmer-Cetus, CAl. After an initial incubation at 94°C for 5 min, PCR was carried out for 30 cycles v consisting of 94°C for 30 seconds, 55°C for 1 min, an d 72°C for 2 min on an Omnigene Temperature Cycler (Hybaid, UK). Cycle Sequencing PCR fragments were resolved on 1.5% low melting Figure 1. IBS is inherited in an autosomal dominant fashion. Pedi­ point agarose gels and excised bands purified usin g Gelase (Epicentre, USA). grees ofIBS families under stud y. ArrolVs, probands in each family. Family 1 Approximately 50 fmol of purified template DNA was sequcnced by the shows clear autosomal dominant Mendelian inheritance; family 2 is a spo­ DS-Cycle Sequencing system (BRL, Bethesda, MD) using a [32P) end-la­ radic case. Individuals arc numbered by standard genetics notation; ., those beled internal primer, K2-7 (5' GTTGAA TGACCTGGAGGAGG 3',+ which took part in this study. strand). Cyele sequencing was performed using the following program: 20 cycles consisti ng of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 mill; followed by 10 cycles consistin g of 94 ° C for 30 scconds, 72 ° C for 1 min. Sequencing ladders were resolved on standard 6% polyacrylamide den­ mis associated with cell lysis was not apparent. These altered cells aturing gels. could occur focally in groups rather than diffusely throughout the upper epidermis. Keratohyalin granules were often small and indis­ Mutation Screening by Restriction Fragment Length Polymor­ tinct. phism (RFLP) The mutation found in both families destroys a recogni­ In some sections, the stratum corneum had become detached just tion site for the restriction enzyme Mull. Due to the close proximiry of intron VII, it was necessary to sequence part of this intron to design a primer above its normal junction with the granular layer. for screening purposes. This was carried out by cycle sequencing as above Electron microscopy revealed a structurally normal basal and lo­ with primer K2-7 (above). For screening, a 163-bp fragment containing the wermost suprabasal layer. Bundling or clumping of the tonofila­ mutation was PCR amplified using primers K2-7 and K2-14 (5' TTC CCA ments was evident in the mid-spinous layer and became more GTG CCC ACA CCT G 3', -strand, intron VII). Primer K.2- 14 contains a m arked in the the upper spinous and lower granular layer (Fig 2a) . I-bp mismatch to remove an unwanted Mull site that would mask the The filament clumps tended to be dispersed more in the cell periph­ mutated site in the assay. PCR was carried out in 25/11 volumes in micro­ ery than around the nuclei; much of the cytoplasm around the cells plates using the conditions described above except that primer K2-14 was edematous by light microscopy was fil ament free and contained end-labeled with [32P). The following PCR program was used: 30 cycles polyribosomes, mitochondria and, in the granular layer, lamellar consisting of 94°C for 30 seconds, 55°C for 1 min, 72°C for 1 min. After PCR, 5 /11 of 6 X Mull buffer containing 6 mM spermidine was added. granules. Keratohyalin was associated with the clumps and also Following incubation at 60°C for 10 min, samples were cooled and incu­ occurred as small round granules associated with polyribosomes bated for 4 h at 37°C with 1 U of Mull (New England Biolabs). An addi­ (Fig 2a). In normal spinous or granular epidermal keratinocytes the tional Mill! site in the fragment is located 6 bp 5' from the mutated site so tonofilam ents were often found in loose bundles (Fig 2b) but never that digests were denatured and resolved on 6% sequencing gels. Digestion formed the tight clumps as seen in IBS skin. of normal alleles gives rise to a 34-bp fragment. Digestion of the mutant allele gives rise to a 40-bp fragment. Affected heterozygotes produce 34- and IBS Patients Axe Heterozygous for a K2e Mutation A 1.5-kb 40-bp fragments. fragment of the K2e gene was amplified from genomic DNA sam­ ples using primers K2-5 and K2-6. This fragment contains all of RESULTS exon7, which encodes part of the 2B domain and the helix termina­ IBS Is Clinically Distinct from BCIE The proband in family 1 tion peptide of K2e, intron VlI, and a small portion of exon 8 is a 10-year-old girl (Fig 1) who was born with normal skin. She encoding the V2 domain. 256 bp of this peR fragment are coding has never been erythrodermic. Blisters developed within a few sequence and so the deduced size of intron VII is about 1.25 kb, monthsof age, mainly localized to the hands and legs, precipitated about twice the size of the equi valent intron in Kl. However, the by phYSical trauma. From the age of 6 months, keratotic li chenifica­ position of the intron was determined by direct sequencing of the tion was noted over the extensor aspects of the elbows and knees, the peR product and is completely conserved, in keeping with the wrists, and the periumbilical area. On the upper, outer aspects of the other type II keratins [14].lntron splice sites were found to conform arms, superficial erosion of skin was seen, a phenomenon known as to the reported consensus sequences (15]. rna use rung or moulting phenomenon. N ails, hair, and oral mucosa Gel-purified peR products derived from affected and unaffected were normal. There was no palmoplantar keratoderma. Other individuals from IBS families 1 and 2 were directly sequenced using members of the family are affected in five consecutive generations. an internal primer K2-7, located in exon 7 . In five unaffected indi­ The proband in family 2 has a phenotype indistinguishable from viduals analyzed from families 1 and 2 and in four normal, unrelated those affected in family 1. N o other family members are affected. individuals, the coding sequence was found to be identical to the published cDNA sequence, with no polymorphisms or silent muta­ Outer Suprabasal Cells in IBS Contain Abnormal Tonofil­ tions observed. However, all affected individuals analyzed in both aments The microscopic findings were essentially the same in all lBS families (five in total) were found to be heterozygous for a G to

biopsy samples. Light microscopy showed hyperkeratosis with A transition at nucleotide 1510 of the eDNA sequence (GJslOA) as poorly compacted cornified cells; slight or moderate acanthosis and shown in Fig 3a. This produces a change of glutamic acid to lysine papillomatosis; and a minimal inflammatory cell infiltrate in the in codon 493 (E493K) in the highly conserved LLEGEE helix termi­ superficial dermis. Epidermal cells from the mid-spinous layer up to nation m otif and thus is likely to be highly detrimental to keratin and including the granular layer appeared enlarged with a pale filament assembly and/or functional integrity (Fig 3b) . This muta­ edematous or vacuolated cytoplasm. Disintegration of the epider- tion is analogous to those reported in KS causing EBS-DM [16] and VOL. 103. NO.3 SEPTEMBER 1994 MUTATIONS IN KERATIN 2c 279

b

Figure 2. Suprabasal tonofilaments are abnormal in IBS. a) In lBS epidermis. tonolilamcnts (small arrows) appear normal in basal layer (B) and lowest suprabasal layer. but are replaced by abnormal clumps or amorphous bodies (opell arrow) in the mid-spinous layer. Note that the clumps tend to be distributed in the cell periphery. Bar, 2 J.l,m. b) Electron micrograph of normal keratinocytes in upper sp inous layer. The tonolilaments in these cell s, in contrast to lBS skin (a), form loose bundles (arrows) around the nuclei (N). extending peripherally to associate with desmosomes (opelJ arrow). Bar, 2 J.l,m.

a in K 1 causing BCIE [17] thus demonstrating once again the sensi­ that are associated with IBS, a pathologic condition affecting the tivity of this motif to pathologic mutation in keratin molecules. outer layers of the epidermis. The base change observed in two The mutation produces a Mull restriction fragment length poly­ unrelated incidences is consistent with a methylated CpG deamina­ morphism that was used to design a PCR-based screening test. Di­ tion mutation of the 5-methyl-cytosine on the antisense strand at gestion of [32P]-labeled PCR products amplified from normal indi­ position 1510. This results in a CG to CA transition in the sense viduals using primers K2-7 and K2-14 results in a 34-bp band. All strand. Methylated CpG sequences have a higher mutation rate than affected members of both families were found to have an additional other dinucleotides, taking the obvious bias for deleterious muta­ 40-bp band (Fig 4). This test was used to exclude the mutation from tions into account [19]. Here, IBS family 1 has affected members in 50 normal unrelated control samples indicating that this mutation is five generations; however, the affected member of IBS fami ly 2 not a common polymorphism. represents a new mutation causing this disease, the parents having Members of family 2 were genotyped with a number of highly been shown to have a normal K2e sequence. Kremer et al [18] de­ polymorphic markers (data not shown). Normal Mendelian inheri­ scribe two further unrelated Dutch families with identical muta­ tance with no evidence of non-paternity was observed; therefore, it tions. The occurrence of the same mutation in four apparently unre­ is likely that this sporadic case represents a new mutation. lated families including one sporadic case indicates that this is likely to be a common mutation causing rBS. DISCUSSION IBS Ultrastructural Changes Are Consistent with Pheno­ The Observed K2e Mutation May Be a Common Cause of type At the electronmicroscopic level, IBS is a disorder character­ ms Here we describe (with Kremer et al [18]) mutations in K2e ized by filament aggregation in the upper suprabasal cells leading to 280 McLEAN ET AL THE J O URNAL OF INVESTIGATIVE DERMATOLOGY

Family 1

ISS ISS Normal Normal ATe GAT C GAT C GAT C

Family 2

ISS Normal Normal GAT C GAT C GAT C

a MnlI MnlI Normal 1486 ACC TAC CGC AAA CTG . C~G GAGIGGCIGAG gAG 1515 485 T Y R K L LE G E E 494

MnlI IBS 1486 ACC TAC CGC AAA CTG C ~G GAG\GGC AAG GAG 1515 485 T Y R K L LEG K E 494 b

Figure 3. DNA Sequencing for Families 1 and 2. a) IBS individuals are heterozygous for a G to A mutation in K2e. Excerpts from DNA sequencing gels showing K2e sequence derived from IBS affected and unaffected individuals in both families 1 and 2. IBS individuals are shown to be heterozygous for a G to A transition corresponding to nucleotide 1510 of the cDNA sequence (arrows). b) The IDS mutation in both fami lies results in an E to K transition. DNA sequence and ded uced protein sequence in the region of the reported mutation showing the loss of an MIIII restriction site (GGAG) in the mutant allele. The LLEGEE motif is most highly conserved within the various intermediate filam ent proteins.

hyperkeratosis and shedding of the stratum corneum. There are a mis and also tended to be distributed more peripherally in the cells. number of ultrastructural features that are distinct from BClE. The clinically superficial erosions or "moulting" probab ly results Firstl y, frank lysis of epidermal cells is not a prominent feature. from separation of the stratum corneum at a low level. This could be Secondly, tonofilament clumps were not seen in the lower epider- due to abnormal structure of horny cell s as reported in BCIE [20]. VOL. 103, NO.3 SEPTEMBER 1994 MUTATIONS [N KERAT[N 2e 281

FAMILY I FAMlLY2 Thallks to Jolm McGratll, St. JohtlS ItlSitLIte oJDermatology alld Adrian Heaga rty, I rl------' Departmeut of Dermatology, North Staffordshire Royal Illfirma,-y, Stoke-oll-Treut, III-3 IV -6 III-2 IV -2 V-I V -2 I-I 1-2 II-I UK Jor ciillical assistallce; Trish Doppillg-Hepeustal alld lOll Palmer Jo r tec/mica l help; alld Hatmie Kremer, Departmellt oj Hllmall Gellelics, Ulli1lersity Hospital Nijmegetl Jor sharillg data alld Jor helpflll disClissiolls. Thallks to Dot M echall alld Dallid Baty, Gelleties Departmellt, Nillcwells Hospilal, DlIlldeeJor DNA process­ illg. This work was supported by gra llisJrom The Wel/co",e Trusl (037444 /A/93 / Z: EBL, RAJE, mId IML), Callcer Research Campaigll (SP2060, EBL), Mllir­ 40bp ... head Trllst (RAJE), alld Dystrophic Epidermolysis BIII/osa Resea rch Associalioll (EBL). 34bp ... REFERENCES

1. McKusick VA: OMIM (O"-/i,,e Mwdclia" 1"herila"cc i" Mall). Johns Hopkins University, .Baltimore 2. Siemens HW: Dichtung und Wahrheit uber die die "ichthyosis bullosa", mit Bemerkungen zur Systematik der Epidermolysen. Arch Dermalol Syph (Berl) 175:590-608, 1937 3. Traupe H , Kolde G, Hamm H, Happle 11.: Ichthyosis bullosa ofSeimens: a unique type of epidermolytic hyperkeratosis.] Alii Acad Derlllalo/14: 1000 - 1005, 1986 4. Traupe H: The ichlhyoses. A gllide 1o e1i"i,al diag"osis, ge"clic colltlSclli"g, a"d Iherapy. Springer-Verlag, Berlin, 1989 Figure 4. IBS individuals are heterozygous for a M"R polymor­ 5. Steijlen PM, Perret CM, Schuurmans Stekhoven JH, Ruiter DJ. Happle R: Ich­ phisttl. Restriaion fragl11en~ ~l1alysis of M,liI digested PCR products de­ thyosis bullosa of Siemens: further delineation of phenotype. Arch Derlllalol Res rived from individuals 111 families 1 and 2. Normal allele gives rISe to a 34-bp 282:1-5, 1990 fragment. 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Steinert PM, Marekov LN , Fraser RDB, Parry DAD: Keratin intennediate fila­ the rod ends are really involved in overlap interactions between ment structure: crosslinking studies yield quantitative information on molecu­ dimers or tetramers, these interactions would explain why muta­ lar dimensions and mechanism of assembly.) Mol Bioi 230:436-452. 1993 25. Geisler N , Heimbu.rg T, SchunemannJ, Weber K: Peptides from the conserved tions in these parts of the protein are severe in their effects. How­ ends of the rod domain ofdesmin disassemble intemlediate filaments and re veal ever, the evidence for this is largely circumstantial at present and unexpected structural fcannes: a circular dichroism, fourier transfrol11 infrared, awaits direct experimental proof. and electron microscopic study.) Slmtl BioI 110:205-214, 1993