Keratin 17 Mutations Cause Either Steatocystoma Multiplex Or Pachyonychia Congenita Type 2

Home , Keratin 16, Steatocystoma multiplex

British Journal of Dermatology 1998; 139: 475–480.

Keratin 17 mutations cause either steatocystoma multiplex or pachyonychia congenita type 2

S.P.COVELLO, F.J.D.SMITH, J.H.SILLEVIS SMITT,* A.S.PALLER,† C.S.MUNRO,‡ M.F.JONKMAN,§ J.UITTO¶ AND W.H.I.McLEAN Epithelial Genetics Group, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 South 10th Street, Philadelphia, PA 19107, U.S.A. *Department of Dermatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands †Division of Dermatology, Children’s Memorial Hospital, Chicago, IL 60614, U.S.A. ‡Department of Dermatology, Southern General Hospital, Glasgow G51 4TF, U.K. §Department of Dermatology, University Hospital, Hanzeplein 1, NL-9700 Groningen, The Netherlands ¶Department of Dermatology and Cutaneous Biology, Jefferson Institute for Molecular Medicine, 233 South 10th Street, Philadelphia, PA 19107, U.S.A. Accepted for publication 8 April 1998

Summary Pachyonychia congenita type 2 (PC-2; Jackson–Lawler syndrome) is an autosomal dominant disorder characterized by hypertrophic nail dystrophy, mild focal keratoderma, multiple pilosebaceous cysts and other features of ectodermal dysplasia. Keratin 17 (K17) is a differentiation-speciﬁc keratin expressed in the nail bed, hair follicle, sebaceous gland and other epidermal appendages. Previously, we have demonstrated that PC-2 is caused by mutations in K17 and that similar mutations in this gene can present as steatocystoma multiplex with little or no nail dystrophy. Here, we describe three unrelated kindreds carrying K17 mutations. Two of these families have identical missense mutations (R94C) in the 1A domain of K17. However, while affected members of one kindred have the classical features of PC-2, affected persons in the other family have the steatocystoma multiplex phenotype. In a third family with PC-2, mutation N92S was detected, bringing the total number of distinct mutations reported in K17 thus far to 11. These results demonstrate that K17 mutations commonly underlie both PC-2 and steatocystoma multiplex and that the alternate phenotypes which arise from these genetic lesions in K17 are independent of the speciﬁc mutation involved.

Keratins form the intermediate filament cytoskeleton of Pathogenic mutations in human keratin disorders most epithelial cells. In humans, there are many different often occur in the helix boundary motifs, further forms of epithelial cell, each with a specific pattern of attesting to the structural importance of these keratin expression according to tissue context and state sequences.5–7 of differentiation.1 Specific pairs of type I and type II Pachyonychia congenita (PC) is a group of autosomal keratins form obligate heterodimers which undergo dominantly inherited diseases characterized by hyper- additional polymerization steps to produce 10 nm inter- trophic nail dystrophy accompanied by varying features mediate filaments. The higher order assembly of keratin of ectodermal dysplasia.8 Many cases of PC are accom- dimers is poorly understood. However, the helix bound- panied by variable focal keratoderma and follicular ary motifs, short sequences located at the start and end keratosis, but two main clinical syndromes have been of the central coiled-coil rod domain, have been impli- distinguished on the basis of other features. Gorlin et al.9 cated in molecular overlap interactions in filament designated the form with oral leucokeratosis10 PC type assembly by means of their remarkable degree of evolu- 1 (PC-1), and that with multiple epidermal cysts11 PC tionary conservation,2 as determined by in vitro muta- type 2 (PC-2). In PC-2 focal keratoderma may be present genesis studies3 and chemical cross-linking analysis.4 in a milder form than in PC-1, but there are more varied ectodermal defects including natal teeth, pili torti,

Correspondence: Dr W.H.Irwin McLean. angular chelosis and hoarseness. Recently, the E-mail: [email protected] molecular basis for these two forms of PC was found

᭧ 1998 British Association of Dermatologists 475 476 S.P.COVELLO et al. to be mutations in keratins which are differentially fragment was observed only in affected members of expressed in epithelial cells of the nail bed and the families 1 and 2 and not in 50 normal unrelated other ectodermal structures affected in these diseases. controls. PC-1 was found to be caused by heterozygous mutations in keratin 16 (K16)12 or in its expression partner, keratin 6a (K6a).13 Similar genetic lesions in keratin Results 17 (K17) were found to cause PC-2.12 More recently, Case reports mutations in K17 have been demonstrated in families presenting as steatocystoma multiplex.14 Further exam- The pedigrees of the three families studied, designated 1, ination of the latter kindreds revealed subtle nail 2 and 3, each with hallmarks of an autosomal changes in some affected individuals. Similarly, K16 dominant mode of inheritance, are shown in Fig. 1. mutations have been identified in patients with focal non-epidermolytic palmoplantar keratoderma, having Family 1. The proband is a 39-year-old American minor nail changes which do not resemble pachyony- caucasian woman who had a history of subcutaneous chia.15 By implication, K6a mutations may also be yellow nodules on the flexor surfaces of the arms, expected to cause this phenotype, although no muta- abdomen and legs since puberty. She also had thickened tions of this type are known. No type II expression nails of the feet. Histological examination of a biopsied partner of K17 has been reported and to date mutations cyst revealed that this was a steatocyst (data not shown) only in this protein have been described in PC-2 or and a diagnosis of Jackson–Lawler form of PC was steatocystoma multiplex families. made. Her son, aged 12 years, had similar nail dystro- Here, we demonstrate mutations in the helix initia- phy (Fig. 2a) and was also seen to be developing tion motif of K17 in affected members of three unrelated steatocystoma multiplex (Fig. 2b). families. Two of these kindreds have the hallmarks of PC-2 and one family has steatocystoma multiplex as the Family 2. The index case is a 41-year-old Dutch cauca- only phenotype. sian woman who presented at an out-patient clinic because of what she described as ‘acne present from Materials and methods puberty’. The number of lesions had increased with age; they were also present on the abdomen, arms and legs. Genomic DNA was extracted from peripheral blood Of great concern to her was the fact that her daughter of lymphocytes by standard methods. Exon 1 of the 4 years and son of 11 years of age were developing KRT17A gene was amplified by polymerase chain similar skin problems. On examination, we noted multi- reaction (PCR) and directly sequenced with forward ple nodules of varying diameter spread over the face, and reverse internal primers as described previously.12 neck, trunk and extremities (Fig. 2c). These were most This PCR is specific for the functional K17 gene.16 This widespread in the mother (not shown). None of the PCR reaction is particularly sensitive to magnesium affected persons showed any nail changes (Fig. 2d), or concentration and was found to perform best with any other skin, hair or mucosal abnormalities.

1 mmol/L MgCl2; however, magnesium titration is recommended. The K17 mutation N92S creates a Family 3. The proband in family 3 was an 18-year-old new DdeI site which was used to conﬁrm the mutation British caucasian who presented with rough skin due to in the affected individuals and also to exclude the follicular keratoses, and thickened nails and plantar mutation from 50 normal unrelated individuals. PCR skin. Although she did not have clinically obvious was performed using primers K17p8 and K17p10,12 digested with DdeI without further puriﬁcation, and the digests were analysed on 1·5% agarose/TBE minigels. Mutation R94C destroys an AciI site. Nested PCR was performed initially with primer K17p8 and K17p10 (see above) and subsequently with primers K17p3 and K17p4.12 The secondary PCR products were exhaus- tively digested overnight at 37 ЊC with an excess of AciI Figure 1. Pedigrees of the pachyonychia congenita type 2 (PC-2) and to ensure complete cutting. Digests were analysed on steatocystoma multiplex families. The arrow indicates the proband in 3·5% NuSieve agarose/TBE minigels. An uncut each kindred.

᭧ 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 475–480 K17 MUTATIONS IN PACHYONYCHIA AND STEATOCYSTOMA MULTIPLEX 477

Figure 2. Clinical features of the patients. (a) Pachyonychia of the toenails seen in individual II1 in family 1, particularly prominent on the great toes. (b) Milia developing on the face of person II1 in family 1, typical of individuals with pachyonychia congenita type 2 at puberty. (c) The back of individual II2 in steatocystoma multiplex family 2, showing prominent cysts, typical of all affected persons in the three kindreds studied. (d) The fingernails of person II2 in family 2 are seen to be completely normal, as were all finger and toenails in all members of this kindred (not shown). cysts, and had mild hyperkeratosis of the buccal domain of K17 in all three families, as shown in Figure mucosa, other family members had abnormal nails, 3. Although unrelated and with differing phenotypes, blistering of feet and multiple cysts. The proband’s families 1 and 2 had the identical pyrimidine transition mother was examined. She gave a history of painful mutation 428C T, predicting amino acid change keratoses and blistering on the feet, with thickened R94C, at the tenth→ amino acid of the helix initiation nails, yellowish cysts on the trunk and limbs, and motif. This mutation abolishes a recognition site for the recurrent flexural abscesses. She also had multiple restriction enzyme AciI, which was used to confirm the milia. Histological examination of an excised lesion mutation in the affected individuals of both families (not from the labium majus revealed an epidermoid cyst shown). This test was also used to exclude mutations in (data not shown). This family has been previously this codon from 50 normal unrelated persons, as reported as having PC with hidradenitis suppurativa.17 described previously.14 This is the first report of this particular mutation in K17. In family 3, the heterozygous purine transition muta- Mutation analysis and confirmation tion 423A G was detected in the proband (Fig. 3), Exon 1 of the K17 gene was amplified from affected predicting amino→ acid substitution N92S, at the eighth persons and normal unrelated controls using PCR residue of the helix initiation sequence. The same primers and conditions which we have previously mutation was detected previously in four unrelated shown to be specific for the functional K17 gene PC-2 families.14 This sequence alteration creates a (KRT17A) and which do not amplify either of the two new recognition site for the restriction enzyme DdeI, K17 pseudogenes (␺KRT17B and ␺KRT17C).12,14 which was used to confirm the mutation in the proband Direct automated sequencing of these PCR products and exclude it from 50 normal individuals, as shown revealed heterozygous missense mutations in the 1A previously.14

᭧ 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 475–480 478 S.P.COVELLO et al.

Discussion Keratin 17 mutations in pachyonychia and steatocystoma multiplex families By direct sequencing of K17 genomic PCR products, we have identified missense mutations in three unrelated pedigrees, making a total of 11 K17 mutations in PC-2 and steatocystoma multiplex families published to date.12,14 Mutation R94C was detected in two unrelated families and is a novel mutation in K17, although an analogous mutation has been reported in other keratins.7 This mutation is likely to have arisen via the 50 methyl-cytosine deamination mechanism,18 which accounts for the prevalence of mutations in this codon of type I keratins.7,19 Two possible arginine substitu- tions can result from deamination mutation of this codon, and the other one, R94H, has been reported in K17 previously.14 The mutation N92S has been reported previously by this group in a further four PC- 2 kindreds.14 All three mutations occur in the 1A domain of the K17 polypeptide, within the helix initiation motif, the site where all reported mutations in this gene occur.7 This sequence is thought to be involved in end-to-end overlap interactions in the assembly of keratin filaments,3,4 and is the site where most mutations in type I keratins have been found.5 Therefore, these mutations are predicted to be detrimental to the keratin cytoskeleton, producing cell fragility and hyperkeratosis in tissues expressing K17. These epithelia include the nail bed, hair follicle and sebaceous gland,16,20 explain- ing the phenotypes of nail dystrophy and steatocystoma Figure 3. Direct sequencing of genomic polymerase chain reaction multiplex seen in these patients. products derived from exon 1 of the KRT17A gene, which encodes the keratin 17 (K17) polypeptide,16 as described previously.12,14 Nucleo- tides 416–436 inclusive of the normal K17 sequence are shown (top The nature of cysts associated with keratin 17 mutation panel), encoding part of the highly conserved helix initiation motif of K17. Heterozygous missense mutation 428C T (arrow), which pre- Although usually referred to as steatocystoma multi- dicts the amino acid change R94C, was found→ in affected persons in plex, the heterogeneous nature of the cysts in PC-2 has families 2 (centre panel) and 1 (not shown). Heterozygous missense caused confusion. Reviewing previous reports of either mutation 423A G (arrow) was found in the proband of family 3 (lower panel), predicting→ the amino acid change N92S. These missense epidermoid cysts or steatocysts, Clementi et al. suggested mutations which occur in a part of the K17 protein which is known to that the two types might be genetically distinct forms of be involved in molecular overlap interactions in the formation of PC-2.21 However, within a single large pedigree,22 and keratin filaments, are predicted to be highly detrimental to the indeed in a single individual, we have critically exam- assembly and/or integrity of the keratin cytoskeleton in cells expressing the mutant polypeptide. ined multiple cysts and found some to be true steatocysts and others keratinous cysts, some of which mutations are highly heterogeneous, but form part of contained vellus hair (Munro CS et al., unpublished the spectrum of follicular hybrid cysts.25 The reasons for data). Milia, flexural abscesses identical to hidradenitis cyst formation are unknown but are presumably related suppurativa17 and scrotal and vulvar cystomatosis also to hyperkeratosis producing infundibular or ductal form part of the syndrome. Eruptive vellus hair cysts occlusion. The histological type of cyst may be deter- and steatocystoma multiplex may be variants of a single mined by the initial state of development of hair or entity,23 and the combination has also been reported sebaceous gland in the pilosebaceous unit, or the site of with PC.24 Thus, the cysts associated with K17 obstruction.

᭧ 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 475–480 K17 MUTATIONS IN PACHYONYCHIA AND STEATOCYSTOMA MULTIPLEX 479

Diagnostic criteria in pachyonychia congenita and steato- modifiers is unknown but is likely to be a combination cystoma multiplex of both genetic and environmental factors. Certainly, trauma is a factor in producing hyperkeratosis in kera- The patients here were diagnosed as PC-2 or steatocys- tinizing disorders and so life-style variables may well toma multiplex on clinical and pathological grounds influence nail dystrophy. In transgenic animals, genetic prior to molecular genetic analysis. However, classifica- background influences the outcome of targeted keratin tion on the basis of clinical features may be misleading mutations,26,27 but the identification of specific acces- in individual cases, as for example in the case of family 3 sory genes in humans is difficult, due to the size of who had no cysts but did have mild oral lesions. pedigrees required to study the segregation of distinct Although the latter are primarily a feature of PC-1, phenotypes. these can be seen in PC-2, reflecting some K17 expres- This study demonstrates that at least some families sion in the oral mucosa. Hoarseness may occur in either with steatocystoma multiplex may have K17 mutations, syndrome, and suggests laryngeal mucosal involve- so that molecular analysis is an option to be considered ment. K17 is presumably also among the keratins for this disorder. PC-2 is considered by some authors to expressed in association with developing teeth, be much less prevalent than PC-1,9,28 and one explana- although how this results in premature eruption is tion for this is the possibility that many K17 mutations not known. We have previously shown14 that some may present as steatocystoma multiplex without cases diagnosed both clinically and histologically as pachyonychia. It should be emphasized, however, that pure steatocystoma multiplex are due to K17 mutation. we have previously examined the K17 gene in sporadic It is likely that K17 mutations are responsible for a cases diagnosed as steatocystoma multiplex or eruptive proportion of cases which otherwise pass clinically as vellus hair cysts without identifying mutations. Thus, steatocystoma multiplex, but in which mild nail there may be other factors, genetic or otherwise, changes in patients or relatives are overlooked. The involved in these epidermal cyst disorders. size of this proportion is unknown but is possibly small, as we have failed to find mutations in other cases of steatocystoma multiplex and of eruptive vellus Acknowledgment hair cysts. We wish to thank Hans-Ju¨rg Alder and his staff, Nucleic Acid Facility, Kimmel Cancer Center, Jefferson Two phenotypes arising from one keratin 17 mutation Medical College, Philadelphia for DNA synthesis and sequencing. This work was supported by grants from Although two of the families examined here share the the Dystrophic Epidermolysis Bullosa Research Asso- same K17 mutation, R94C, these differ in phenotype in ciation (DEBRA), U.K., and the U.S. Public Health that affected members of family 1 have the typical PC-2 Service, National Institutes of Health (grant PO1- nail dystrophy and epidermal cysts, whereas affected AR38923). persons in family 2 have no nail changes and steatocystoma multiplex is the only phenotype (Fig. 2). Pre- viously, we have shown that families with very similar References mutations in K17 could give rise to these similar but 1 Lane EB. Keratins. In: Connective Tissue and its Heritable Disorders. distinct phenotypes, specifically mutations R94H and Molecular, Genetic and Medical Aspects (Royce PM, Steinmann B, N92H in steatocystoma multiplex families and muta- eds). New York: Wiley-Liss Inc., 1993; 237–47. tions N92D, N92S and Y98D in PC-2 kindreds.12,14 2 Quinlan RA, Hutchison CJ, Lane EB. Intermediate Filaments. London: Academic Press, 1994. Phenotypic variation is common in other keratin 3 Letai A, Coulombe PA, Fuchs E. Do the ends justify the means? 7 diseases. From our previous studies of keratin diseases, Proline mutations at the ends of the keratin coiled-coil rod we felt that this is not closely related to genotype, but we segment are more disruptive than internal mutations. J Cell Biol could not rule out the possibility that these two pheno- 1992; 116: 1181–95. 4 Steinert PM, Marekov LN, Fraser RDB, Parry DAD. Keratin inter- types might be related to the specific mutations mediate filament structure: crosslinking studies yield quantitative involved. However, here we see both phenotypes arising information on molecular dimensions and mechanism of assem- from the same mutation, R94C, confirming that the bly. J Mol Biol 1993; 230: 436–52. variable phenotype phenomenon is not due to the 5 Steinert PM, Yang JM, Bale SJ, Compton JG. Concurrence between the molecular overlap regions in keratin intermediate filaments specific K17 mutation alone, but must depend on a and the locations of keratin mutations in genodermatoses. Bio- combination of other factors. The identity of these chem Biophys Res Commun 1993; 197: 840–8.

᭧ 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 475–480 480 S.P.COVELLO et al.

6 Letai A, Coulombe PA, McCormick MB et al. Disease severity human gene encoding cytokeratin 17 and its expression pattern. correlates with position of keratin point mutations in patients Eur J Cell Biol 1992; 59: 127–37. with epidermolysis bullosa simplex. Proc Natl Acad Sci USA 1993; 17 Todd P, Garioch J, Rademaker M et al. Pachyonychia congenita 90: 3197–201. complicated by hidradenatis suppurativa: a family study. Br J 7 Corden LD, McLean WHI. Human keratin diseases: hereditary Dermatol 1990; 123: 663–6. fragility of specific epithelial tissues. Exp Dermatol 1996; 5: 297– 18 Cooper DN, Krawczak M. Human Gene Mutation. Oxford: BIOS 307. Scientific Publishers Ltd, 1993. 8 Stevens HP, Kelsell DP, Bryant SP et al. Linkage of an American 19 McLean WHI, Lane EB. Intermediate filaments in disease. Curr pedigree with palmoplantar keratoderma and malignancy (pal- Opin Cell Biol 1995; 7: 118–25. moplantar ectodermal dysplasia type III) to 17q24. Literature 20 Troyanovsky SM, Guelstein VI, Tchipysheva TA et al. Patterns of survey and proposed updated classification of the keratodermas. expression of K17 in human epithelia: dependency on cell posi- Arch Dermatol 1996; 132: 640–51. tion. J Cell Sci 1989; 93: 419–26. 9 Gorlin RJ, Pindborg JJ, Cohen MM Jr. Syndromes of the Head and 21 Clementi M, Cardini de Stafani E, De Rossi C et al. Pachyonychia Neck. New York: McGraw-Hill, 1976. congenita Jackson-Lawler type: a distinct malformation syn- 10 Jadassohn J, Lewandowsky F. Pachyonychia congenita. In: Jacobs drome. Br J Dermatol 1986; 114: 367–70. Ikonographia Dermatologica. Berlin: Urban and Schwarzenberg, 22 Munro CS, Carter S, Bryce S et al. A gene for pachyonychia 1906; 29. congenita is closely linked to the keratin gene cluster on 17q12- 11 Jackson ADM, Lawler SD. Pachyonychia congenita: a report of six q21. J Med Genet 1994; 31: 675–8. cases in one family. Ann Eugen 1951; 16: 142–6. 23 Keine P, Hauschild A, Christophers E. Eruptive vellus hair cysts 12 McLean WHI, Rugg EL, Lunny DP et al. Keratin 16 and keratin 17 and steatocystoma multiplex. Variants of one entity? Br J Dermatol mutations cause pachyonychia congenita. Nat Genet 1995; 9: 1996; 134: 365–7. 273–8. 24 Moon SE, Lee YS, Youn JI. Eruptive vellus hair cyst and steatocys- 13 Bowden PE, Haley JL, Kansky A et al. Mutation of a type II keratin toma multiplex in a patient with pachyonychia congenita. JAm gene(K6a)in pachyonychiacongenita. NatGenet 1995;10: 363–5. Acad Dermatol 1994; 30: 275–6. 14 Smith FJD, Corden LD, Rugg EL et al. Missense mutations in keratin 25 Requena L, Yus ES. Follicular hybrid cysts—an expanded spec- 17 cause either pachyonychia congenita type 2 or a phenotype trum. Am J Dermatopathol 1991; 13: 228–33. resembling steatocystoma multiplex. J Invest Dermatol 1997; 108: 26 Baribault H, Price J, Miyai K, Oshima RG. Mid-gestational lethality 220–3. in mice lacking keratin 8. Genes Dev 1993; 7: 1191–202. 15 Shamsher MK, Navsaria HA, Stevens HP et al. Novel mutations in 27 Baribault H, Penner J, Iozzo RB, Wilson-Heiner M. Colorectal keratin 16 gene underlie focal non-epidermolytic palmoplantar hyperplasia and inflammation in keratin 8-deficient FVB/N mice. keratoderma (NEPPK) in 2 families. Hum Mol Genet 1995; 4: Genes Dev 1994; 8: 2964–73. 1875–81. 28 McKusick VA. OMIM (On-line Mendelian Inheritance in Man). 16 Troyanovsky SM, Leube RE, Franke WW. Characterization of the Baltimore: Johns Hopkins University, 1997.

᭧ 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 475–480