Am. J. Hum. Genet. 56:577-585, 1995 Epidermolysis Bullosa Simplex: A Keratin 5 Mutation Is a Fully Dominant Allele in Epidermal Cytoskeleton Function Karen Stephens,' Abraham Zlotogorski,7 Lynne Smith,23 Pamela Ehrlich,' Ellen Wijsman,"4 Robert J. Livingston,l and Virginia P. Sybert 2'5'6 Department of Medicine, Divisions of 'Medical Genetics and 2Dermatology, and Departments of 'Biological Structure, 4Biostatistics, and 5Pediatrics, University of Washington, and 6Children's Hospital and Medical Center, Seattle; and 7Department of Dermatology, Hadassah University Hospital, Jerusalem Summary mediately form a coiled-coil heterodimer. The heterodimers are the building blocks for the higher-order interactions To explore the relationship between abnormal keratin necessary for assembly of the 10-nm-diameter KIF (re- molecules, 10-nm intermediate filament (IF) organization, viewed by Steinert 1993). The tissue- and differentiation- and epidermal fragility and blistering, we sought to deter- specific expression of each mutated keratin gene appears mine the functional consequences of homozygosity for a to be the primary determinant of the clinical phenotype in dominant keratin defect. We describe a family with an epidermal blistering disorders. A single mutation in either autosomal dominant skin-blistering disorder, epidermoly- gene encoding the coexpressed basal cell-specific keratins sis bullosa simplex, Koebner subtype (EBS-K), that has a 5 (K5, type II) and 14 (K14, type I) causes at least three novel point mutation, occurring in the keratin 5 gene of the autosomal dominant disorders known as "epider- (KRT5), that predicts the substitution of an evolutionarily molysis bullosa simplex" (EBS) (Bonifas et al. 1991; Cou- conserved lysine by an asparagine residue (K173N). Unlike lombe et al. 1991; Dong et al. 1993; Rugg et al. 1993; previous heterozygous mutations located within the initial Stephens et al. 1993; Chan et al. 1994a), which are charac- segment of domain 1A of keratin molecules, K173N heter- terized primarily by nonscarring intraepidermal blisters re- ozygosity did not result in severe disease or clumping of sulting from basal cell cytolysis (Fine et al. 1991). Muta- keratin filaments. One family member was found to be tions in the suprabasal cell-specific keratins underlie sev- homozygous for the K173N allele, having inherited it from eral autosomal dominant conditions characterized by each of her affected first-cousin parents. Despite a lack of epidermolytic hyperkeratosis with cytolysis of the su- normal keratin 5 molecules, and an effective doubling of prabasal cells of the spinous layers. For example, mutation abnormal molecules, available for heterodimerization with of keratins 1 or 10 (K1, type II; and K10, type I) result in keratin 14 during IF formation, there were no significant the generalized blistering disorder bullous congenital ich- differences in the clinical severity or the ultrastructural thyosiform erythroderma (BCIE; or epidermolytic hyper- organization of the keratin IF cytoskeleton of the homozy- keratosis [EH]; Traupe 1989), while mutation of keratin 9 gous individual. These data demonstrate that the K173N (K9), expressed in the suprabasal cells of sole and palm, mutation behaves as a fully dominant allele and indicate results in the localized blistering disorder epidermolytic pal- that a limited number of abnormal keratin molecules are mar plantar keratoderma (EPPK; Reis et al. 1994; Torchard sufficient to impair cytoskeletal function and elicit epider- et al. 1994). mal fragility and blistering. Identified keratin gene defects, which include point muta- tions and in-frame deletions (reviewed by Compton 1994), Introduction produce abnormal molecules that disrupt heterodimeriza- tion and/or interactions between heterodimers necessary Several disorders of blistering and fragility of the epidermis for KIF assembly/function. Several lines of evidence provide result from defects in the keratin intermediate filament support for dominant-negative effects of keratin mutations. (KIF) cytoskeleton. KIFs of epithelial cells are assembled Disorganized clumps of KIF in basal and suprabasal cells from cytoplasmic type I (acidic) and type II (neutral basic) are hallmarks of Dowling-Meara, the most severe type of keratin polypeptides that individually are unstable and im- EBS (EBS-DM), and of BCIE and EPPK (Traupe 1989; Fine et al. 1991), respectively. Perturbation of KIF networks has also been induced in vitro by synthetic mutant oligo- Received September 28, 1994; accepted for publication December 19, peptides or during transient transfections by bacterially ex- 1994. pressed mutant K5 or K14 (Coulombe et al. 1991; Letai Address for correspondence and reprints: Dr. Karen Stephens, Univer- et al. 1993; Steinert et al. 1993). In addition, basal cell KIF sity of Washington, Division of Medical Genetics, RG-25, 1959 N.E. networks and tissue architecture in the mouse Pacific Street, Seattle, WA 98195. epidermal © 1995 by The American Society of Human Genetics. All rights reserved. were altered by a K14 transgene with a C-terminal deletion 0002-9297/95/5603-0004$02.00 (Vassar et al. 1991). 577 578 Am. J. Hum. Genet. 56:577-585, 1995 The identification of keratin mutations that underlie hu- nant EBS disorder or the ultrastructure of the basal cell man skin-blistering disorders may reveal critical residues KIF network. or domains involved in assembly and/or function of normal KIF and may explain the molecular basis for the clumping Material and Methods of certain abnormal KIFs. In contrast to the histopathology of EBS-DM, basal cell KIFs do not aggregate into clumps Family-Tissue Studies in other EBS disorders, specifically the Koebner subtype Family EB13, identified and published initially as EBS- (EBS-K) with generalized blistering and the Weber-Cock- DM (Hacham-Zadeh et al. 1988), was recontacted, inter- ayne subtype (EBS-WC) with blistering primarily localized viewed, physically examined, and sampled for blood; the to the hands and feet (reviewed by Fine et al. 1991). Al- homozygous status of individual 31 was unknown at time though all three EBS subtypes result from mutations in of exam. Affected family members had nonscarring gener- K5 or K14, the molecular basis for clumping of certain alized blistering with obvious exacerbation during the sum- abnormal KIF molecules is not known. Functional assays mer and with fever, few or no milia, and no tooth involve- suggest that mutations at the ends of the keratin central ment, as reported elsewhere (Hacham-Zadeh et al. 1988). rod domain are the most effective in perturbing KIF organi- Immnortalized cell lines were established by transformation zation (Coulombe et al. 1991; Letai et al. 1993; Steinert et of peripheral lymphocytes with Epstein-Barr virus (Nietzel al. 1993). Further investigations are necessary to determine 1986). From a total of eight individuals, skin biopsies (2 the effects that specific mutated residues or domains and/ mm), taken from the upper outer arm after rubbing or, in or altered ratios of defective to normal peptides have on one case, without rubbing, were analyzed (see Results). KIF organization and function. Skin biopsies were transported in glutaraldehyde and were In an attempt to identify an individual homozygous processed for electron microscopy as described elsewhere for a keratin mutation, we have studied a large family (Smith and Sybert 1990). with a consanguineous mating between two related indi- viduals with dominant EBS. The phenotype of an indi- Linkage Studies vidual homozygous for a defective keratin would reveal For genotyping, Southern blots were prepared and hy- the functional consequences of effectively doubling the bridized as described elsewhere (Kayes et al. 1992), to number of abnormal keratin molecules available for het- probes pEFD33.2 (D12S14) and pCMM86 (D17S74). erodimer formation, in a background of no normal mol- LOD scores were calculated with the computer program ecules. Although individuals homozygous for mutations LIPED (Ott 1976) modified to handle large numbers of for dominant human disease can provide insight into alleles (Schellenberg et al. 1992). To reduce computation pathogenesis, they are rarely encountered (Wilkie 1994). time, sampled but unaffected individuals in consanguineous Examples of disorders in which homozyogotes were marriages were assumed to be unrelated to the rest of the found to have more severe disease than did their hetero- pedigree. Analyses were done under the assumption that zygous relatives include thalassemia (Weatherall 1994), the disease locus and marker loci were in linkage equilib- familial hypercholesterolemia (Hobbs et al. 1992), rium. The EBS disease allele was assumed to have a fre- Charcot-Marie-Tooth syndrome (Lupski et al. 1991), quency of .005. Allele frequencies used for the marker loci and achondroplasia (Shiang et al. 1994). Fewer domi- were estimated from the observed alleles in the pedigree, nant disorders in which homozygotes and heterozygotes because frequency information was unavailable for a repre- are affected to a similar degree have been reported, per- sentative control population. It should be noted that, in the haps because clinical recognition of homozygosity is pre- case of G531C, this most likely overestimates the frequency cluded and homozygosity can only be identified by mo- of the mutant allele. Analysis with a low allele frequency lecular studies. Mutational and/or linkage analyses have of .0001 for the G521C mutation gave essentially the same