A mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2

A mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2
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  ©  1998 Oxford University Press  1143–1148  Human Molecular Genetics, 1998, Vol. 7, No. 7  A mutation in human keratin K6b produces aphenocopy of the K17 disorder pachyonychiacongenita type 2 Frances J. D. Smith 1 , Marcel F. Jonkman 2 , Harry van Goor 3 , Carrie M. Coleman 1 , Seana P. Covello 1 , Jouni Uitto 1  and W. H. Irwin McLean 1, * 1 Epithelial Genetics Group, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233South 10th Street, Philadelphia, PA 19107, USA, 2 Department of Dermatology and 3 Department of Pathology,University Hospital, Hanzeplein 1, NL-9700 Groningen, The Netherlands Received February 17, 1998; Revised and Accepted March 12, 1998 Type I and type II keratins form the heteropolymericintermediate filament cytoskeleton, which is the mainstress-bearing structure within epithelial cells.Pachyonychia congenita (PC) is a group of autosomaldominant disorders whose most prominent phenotypeis hypertrophic nail dystrophy accompanied by otherfeatures of ectodermal dysplasia. It has been shownpreviously that mutations in either K16 or K6a, whichform a keratin expression pair, produce the PC-1 variant(MIM 184510). Mutations in K17 alone, an unpairedaccessory keratin, result in the PC-2 phenotype (MIM184500). Here, we describe a family with PC-2 in whichthe K17 locus on 17q was excluded and linkage to thetype II keratin locus on 12q was obtained ( Z  max  3.31 at θ  = 0). Mutation analysis of candidate keratins revealedthe first reported missense mutation in K6b, implyingthat this keratin is the previously unknown expressionpartner of K17, analogous to the K6a/K16 pair.Co-expression of these genes was confirmed by in situ  hybridization and immunohistochemical staining.These results reveal the hitherto unknown role of theK6b isoform in epithelial biology, as well as geneticheterogeneity in PC-2.INTRODUCTION Keratins are a multigene family of heteropolymeric intermediatefilament proteins which are expressed in characteristic type I/typeII expression pairs in specific epithelial tissues (1,2). Mutations which either (i) affect the assembly of or (ii) cause completeabsence of keratin filaments result in diseases characterized byfragility of the particular epithelial cells expressing the mutantprotein (3,4). Defects in 16 keratins are now associated with human diseases, the most recent additions being defects incorneal keratins K3 and K12 causing Meesmann’s cornealdystrophy (5), trichocyte (hair) keratins hHb6 or hHb1 in the hairdisorder monilethrix (6,7) and the association of a mutation in simple epithelial keratin K18 with cryptogenic cirrhosis (8).Pachyonychia congenita (PC) is a group of largely autosomaldominant genodermatoses characterized by hypertrophic naildystrophy accompanied by varying features of ectodermaldysplasia (9). There are two main clinical subtypes. TheJadassohn–Lewandowsky form (PC-1), where nail dystrophy isaccompanied by focal palmoplantar keratoderma and oralleukokeratosis, is caused by mutations in keratin K16 (10) or itsexpression partner K6a (11). In the Jackson–Lawler form (PC-2)there is minimal oral involvement, milder keratoderma andmultiple steatocystomas are a major clinical feature. Steato-cystoma, also known as eruptive vellus hair cyst, is a cystichamartoma lined by sebaceous ductal epithelium (12). We havepreviously shown that PC-2 can be caused by mutations in K17(10,13) and that some families carrying K17 mutations have steatocystomas with little or no nail changes (13,14), a phenotype known as steatocystoma multiplex. Similarly, focal keratodermawithout nail changes can result from K16 mutations (15) and, byimplication, K6a mutations.K17 is a type I keratin expressed in a number of epidermalappendages, such as nail bed, hair follicle, sebaceous gland andother structures (16,17). No specific type II expression partner for K17 has been reported and, to date, we have found K17 mutationsin 11 unrelated PC-2 or steatocystoma families (10,13,14). Here, we report the first PC-2 kindred where the causative mutationresides in the type II keratin K6b, revealing genetic heterogeneityin PC-2 and suggesting that this type II keratin is the expressionpartner of K17. Co-expression of these genes in differentiatedepithelia was confirmed experimentally. RESULTS Clinical findings and linkage analysis A pachyonychia family was identified with the hallmarks of autosomal dominant inheritance (Fig. 1) and the constellation of clinical features typical of the PC-2 variant (Fig. 2). In particular,affected individuals had multiple steatocystomas with the histo- *To whom correspondence should be addressed. Tel: +1 215 503 3241; Fax: +1 215 923 9354; Email: mclean2@jeflin.tju.edu     Human Molecular Genetics, 1998, Vol. 7, No. 7  1144 Figure 1.  Pedigree of the pachyonychia congenita type 2 family studied, exhibiting male-to-male transmission, consistent with autosomal dominant inheritance.Asterisks indicate persons from whom DNA was obtained, arrow indicates proband. logically typical crenulated lining and some lesions containingvellus hairs in the lumen, a key diagnostic feature of PC-2 (datanot shown). Exon 1 of the functional K17 gene ( KRT17A ) wasamplified using conditions we have previously reported which donot amplify the two K17   pseudogenes (10). Direct sequencing of this fragment produced normal sequence identical to thatpublished for K17 (17). To circumvent pseudogene contamina-tion, we used cDNA derived from epidermal keratinocytescultured from the skin of the proband to PCR amplify the entireK17 coding sequence. Full-length sequencing again revealed nomutations. DNA was obtained from additional family membersas shown in Figure 1 and linkage analysis with informativemarker D17S800 in the type I keratin cluster on 17q excluded thisas the disease locus. However, linkage was obtained to markerD12S1651 in the type II keratin cluster on 12q, with a lod scoreof  Z  max  3.31 at θ  = 0. Candidate gene screening No type II keratin was known to have the same pattern of expression as K17; however, a clue was taken from the recentreport of cloning six or more human genes encoding isoforms of keratin K6 (18). In this report, the close similarity in the codingsequences of these isoforms precluded detailed analysis of theexpression patterns of these keratins. However, the representationin cDNA libraries of the two major expressed forms (K6a andK6b) was found to differ between different tissues of srcin.Specifically, the K6b isoform was more common in scalpepidermis, relative to epidermal sites, where there are fewer hairfollicles (18). K17 is most strongly expressed in the sebaceousgland and duct which accompany hair follicles (16,17), thus explaining the hyperplastic steatocystomas seen in PC-2 (10, 13). Therefore, we postulated that K6b might be the partner of K17and harbor the mutation in family D.Primers and PCR conditions were developed to specificallyamplify the full-length K6b isoform from keratinocyte cDNA,taking advantage of the fact that the 3 ′ -untranslated region (UTR)has a number of fairly specific sequence sites (18). Sequencing of this fragment derived from the proband keratinocyte cDNArevealed a heterozygous missense mutation 1459G → A, abolish-ing a  Bse RI restriction site and predicting the amino acid changeE472K in the conserved helix termination motif of K6b (Fig. 3).A specific genomic PCR was developed for this region of the K6bgene and by  Bse RI digestion of this fragment, the mutation wasfound to co-segregate with the disease in family D and wasexcluded from 50 unrelated normal individuals. This mutationconforms to the CpG deamination model (19) and the analogousmutation has been seen in a number of other type II keratins wherethis sequence is conserved (4–6). Co-expression of K6b and K17 The close similarity in phenotype between PC-2 patients carryingK17 mutations and the family seen here with a K6b mutationstrongly implies co-expression of these keratins in differentiatedepithelia and so we set out to prove this hypothesis. No antibodiesare known which can distinguish the K6a and K6b isoforms;however, we identified a 373 bp sequence in the 3 ′ -UTR of theK6b mRNA which is poorly conserved in K6a (45% identity).This fragment of K6b was found to only hybridize with itself andnot the equivalent K6a UTR sequence on Southern blots, asshown in Figure 4, demonstrating its specificity.  In situ  hybridiza-tion using the specific K6b UTR probe showed that K6b isexpressed in the luminal cell layers of sebaceous ducts (Fig. 5A),where it co-localized with K17 protein, visualized by immuno-histochemical staining (Fig. 5B). K6b was not expressed inepidermis, in sebaceous acini or in follicular epithelium. Similar-ly, K17 was not expressed in epidermis or in sebaceous acini, but,in contrast to K6b, was present in deeper parts of the follicularepithelium at the level of the istmus and the stem (data notshown). DISCUSSION Here, we have identified for the first time a second gene forkeratin PC-2, K6b, and have shown that a heterozygous missensemutation E472K co-segregates with the disease in a large Dutchkindred. Thus, this disorder is genetically heterogeneous and canbe caused by mutations in either protein of a keratin pair (4). Themutation was detected in the helix termination motif of the K6b  1145  Nucleic Acids Research, 1994, Vol. 22, No. 1 Human Molecular Genetics, 1998, Vol. 7, No. 7   1145 Figure 2.  Clinical features of pachyonychia congenita type 2 observed in theproband. ( a ) Focal palmoplantar keratoderma was observed on the pressurepoints of the feet. Similar focal palmar callosities were seen in some affectedpersons in the family. ( b ) Hypertrophic nail dystrophy (pachyonychia) was seenaffecting all fingernails and toenails. ( c ) Steatocystomas, observed here ontrunk skin, were widespread in post-pubescent affected individuals, typical of PC-2. ( d ) Mild lingual hyperkeratosis was observed on the margins of thetongue. Oral leukokeratosis is prominent in PC-1, since K16 and K6a arestrongly expressed in these epithelia, but is not commonly observed in PC-2.These observations indicate that expression of K6b, like K17, is topographical-ly restricted in oral and lingual epithelia. polypeptide, a short sequence which marks the end of the coiledcoil rod domain. This motif has been implicated in molecularoverlap interactions in the higher order polymerization of keratinheterodimers by virtue of its remarkable degree of evolutionaryconservation (1), the results of in vitro  mutagenesis studies (20)and chemical cross-linking analysis (21). The prevalence of pathogenic mutations in human keratin disorders at the helixboundary motifs further attests to the functional importance of these sites (4,22). The finding that K17 has an expression partner is surprising.The fact that the K6 isoforms only differ by seven isolated aminoacid substitutions (18) has precluded their distinction usingantibodies, so that this pairing had not been identified until now.In general, there is a tendency for mutations to occur more oftenin the type I protein of a keratin pair (4). This is reflected here bythe fact that in 12 unrelated PC-2 families we have studied wedetected 11 K17 mutations before finding the complementaryK6b defect. The presence of a conserved CpG dinucleotide in the1A domain of type I keratins only partly explains this skewing of mutations.The fact that this mutation in K6b produces a phenocopy of theK17 mutant phenotype implies that these keratins form a hithertounknown expression pair. Here, we have confirmed this by acombination of in situ  hybrization with a probe specific for K6band immunohistochemical staining for K17 (Fig. 5). Theexpression of K6b and K17 in the sebaceous duct epitheliummatches with Ackerman’s hypothesis that steatocystomas arecystic hamartomas of the sebaceous duct epithelium (12). Fromthe PC-2 phenotype seen here, we expect that K6b is alsoexpressed in the nail bed and in the epidermis of the palms andsoles. Interestingly, expression of K6b and K17 was found todiffer in deeper regions of the follicular epithelium (data notshown). This may be related to the occurrence of  pili torti  (twistedhair), a minor phenotype seen in PC-2. Although we haveobserved  pili torti  in several families with PC-2 due to mutationsin K17 (10,13,14), this was not observed in the family studied here carrying the K6b mutation, consistent with the reducedfollicular expression of K6b. In the follicular epithelium K17 maypair with a keratin other than K6b, perhaps another K6 isoformwhose tissue distribution and function has yet to be elucidated(18). In view of this, the tissue distributions of the K6 isoformsand K17 require further study.It remains to be seen if the properties of K6a/K17 filamentsdiffer significantly from K6b/K17 or K6b/K16 filaments. Amongthe seven amino acid substitutions between these K6 isoforms,four occur in less well-conserved regions of helix 1 and the H1domain and are predicted to have a modest effect on polymeriz-ation (18). The remaining three substitutions occur in the V1 andV2 domains. The function of keratin variable domains is currentlyunclear, although two types of mutations in the V1 domain pointto either a subtle role in filament assembly or in mediatinginteractions with non-keratin proteins (23,24). Future studies employing the yeast two-hybrid system, substitution of keratinsin epithelial tissues by transgenics and in vitro  biochemical assaysare required to define the specific properties of the manydifferentially expressed keratin filament systems. MATERIALS AND METHODS Genotyping and linkage analysis Microsatellite markers were PCR amplified using 33 P-labeledprimers, analyzed on standard 6% sequencing gels and visualizedby autoradiography. Marker D17S800 was used to exclude thetype I keratin cluster on 17q; marker D12S1651 in the type IIkeratin cluster on 12q was fully informative and showed linkageto the disease. Two-point lod scores were computed using theMLINK algorithm of Linkage v.5.1, assuming a mutant allelefrequency of 0.001 and 100% penetrance. Marker allele fre-quencies were assumed to be equal in the population and in thecase of marker D12S1651 recalculation using a population     Human Molecular Genetics, 1998, Vol. 7, No. 7  1146  Figure 3.  Detection and confirmation of a K6b mutation in affected members of family D. ( a ) Normal cDNA sequence in the region encoding the helix terminationmotif of the K6b polypeptide. ( b ) Analogous region of K6b cDNA sequence derived from the proband, showing heterozygous missense mutation 1459G → A (arrow),which disrupts a  Bse RI restriction enzyme site and is predicted to cause amino acid substitution E472K. ( c )  Bse RI digestion of specific K6b genomic PCR fragmentscontaining the mutation. Lanes 1–3, 5 and 6 represent affected persons from family D, which are heterozygous for the  Bse RI site. Fragments derived from unaffectedfamily members (lanes 4 and 7) showed complete  Bse RI digestion, as did 50 normal unrelated controls (not shown). Figure 4.  Southern analysis of K6a and K6b 3 ′ -UTR fragments. ( A ) Gel usedfor Southern analysis: lane 1, DNA standard type VI (Boehringer); lane 2, 373bp PCR fragment derived from the 3 ′ -UTR region of K6a; lane 3, correspon-ding PCR fragment derived from the 3 ′ -UTR of K6b. ( B ) Southern blot of thegel shown in (A), probed with digoxygenin-labeled   K6b fragment, showingspecificity of this fragment for the K6b isoform. frequency of 50% for the linked allele yielded the same lod scoreof  Z  max  = 3.31 at θ  = 0. Mutation detection and confirmation Primary skin keratinocytes were cultured from punch biopsies inKGM medium (Gibco) by standard methods (25). Poly(A) + mRNA was extracted from the cultured cells using a QuickPrepMicro mRNA Purification Kit (Pharmacia) and reverse tran-scribed using oligo(dT) and AMV reverse transcriptase (Prome-ga). A 2013 bp fragment of the K6b cDNA was amplified usingBoehringer Mannheim High Fidelity buffer containing 1 mMMgCl 2 , 4% dimethylsulfoxide and primers specific for K6bcDNA: K6b.P1 (sense), 5 ′ -CTC CAG CCT CTC ACA CTC TCCTA-3 ′ ; K6b.P2 (antisense), 5 ′ -CTT CTC AGA ATT ATG GCAGAC TCA G-3 ′ . The reactions were subjected to a ‘hot start’ with1 U High Fidelity Enzyme Mix. PCR conditions were 94  C for5 min; 35 cycles of 94  C for 30 s, 55  C for 1 min, 72  C for 2 min;a final extension of 72  C for 5 min. PCR products were purifiedusing a QIAquick PCR purification kit (Qiagen) and directlysequenced with both primers on an ABI 377 automated sequencerusing the ABI PRISM fluorescent dye terminator system (PerkinElmer). Genomic DNA was extracted from whole blood bystandard methods and the mutation was confirmed by directsequencing of K6b-specific genomic PCR products. A fragmentof ∼ 1200 bp was amplified using primers K6b.P5 (sense, 5 ′ -TTTCTC TTC CTT TGC CCT CCT-3 ′ ) and K6b.P2 (antisense,5 ′ -CTT CTC AGA ATT ATG GCA GAC TCA-G-3 ′ ) as above,except that the following conditions were used: 94  C for 5 min;35 cycles of 94  C for 30 s, 55  C for 1 min, 72  C for 2 min; a finalextension of 72  C for 5 min. PCR products were purified andsequenced as above. Mutation E472K abolishes a recognition sitefor the restriction enzyme  Bse RI. Nested PCR was carried outusing primers K6b.P2 and K6b.P5 as above, followed byreamplification using primers K6b.P9 (sense, 5 ′ -GCT GGAAGG GCT GGA GGA TG-3 ′ ) and K6b.p10 (antisense, 5 ′ -TGCTCA GTG CCA GAA CCT TGA A-3 ′ ). The resultant 210 bpfragment, which contains a single  Bse RI site, was exhaustivelydigested with  Bse RI. An uncut band was observed only in digestsderived from affected members of family D. Cloning of a specific K6b probe and Southern analysis A 373 bp fragment from the 3 ′ -UTR of the K6b mRNA wasamplified from normal human keratinocyte cDNA using primersK6B.U1 (+ strand, 5 ′ -GCC CTC ACT TTT CTT CTC ATC  1147   Nucleic Acids Research, 1994, Vol. 22, No. 1 Human Molecular Genetics, 1998, Vol. 7, No. 7   1147  Figure 5.  ( A ) K6b mRNA visualized by non-isotopic in situ  hybridization co-localizes with ( B ) K17 protein in the luminal cell layers of sebaceous glands in theepidermis. The latter was visualized by subsequent immunohistochemical staining of the same tissue section. AA-3 ′ ) and K6B.U2 (– strand, 5 ′ -CAA AGA GAG CAG AGAAAG CAG TG-3 ′ ). Similarly, the corresponding fragment of theK6a 3 ′ -UTR was amplified using primers K6A.U1(+ strand,5 ′ -CTC ACT TCT TCT CTC TCT CTC TAT ACC AT-3 ′ ) andK6A.U2 (– strand, 5 ′ -CAG TGA AGA GCA CAG AAA TCATCA-3 ′ ). These fragments were cloned into the pCR2.1 vector(TA Cloning kit; Invitrogen) and fully sequenced. Clones in bothforward and reverse orientations were selected for synthesis of sense and antisense RNA probes for in situ  hybridization. PCRfragments generated from the K6a and K6b clones were Southernblotted and probed with digoxigenin-labeled K6b fragment. Blotswere washed at 45  C and the signal was detected by anti-digoxigenin Fab fragments conjugated to alkaline phosphatase,with the color substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Boehringer MannheimGmbH).  In situ  hybridization and immunohistochemistry A skin biopsy was obtained from the face of a normal individual.The specimen was fixed in 10% formalin overnight at 37  C andembedded in paraffin. To produce digoxigenin-labeled RNA,sense and antisense K6b cDNA was transcribed in vitro  with T7RNA polymerase in the presence of digoxigenin-UTP accordingto the manufacturer’s protocol (Boehringer Mannheim GmbH).After overnight hybridization at 55  C the sections were treatedwith RNase (Sigma R4875). The hybridized digoxigenin-labeledprobes were immunodetected with anti-digoxigenin Fab frag-ments conjugated to alkaline phosphatase and the boundconjugate was visualized with NBT/BCIP (BoehringerMannheim GmbH). Digital micrographs were taken with a highresolution full color CCD camera (Lumina Systems, West-borough, MA) mounted on a light microscope (Carl Zeiss JenaGmbH). The same section used for K6b in situ  hybridization waswashed with acetone to remove the NBT/BCIP precipitate andprocessed further for K17 detection. For the detection of K17 weused mouse monoclonal antibody E3 (16) as primary antibody,followed by rabbit anti-mouse IgG conjugated to horse radishperoxidase as second antibody (Dako A/S, Denmark). The boundconjugate was visualized with 3-amino-9-ethyl-carbazole in theperoxidase reaction. ACKNOWLEDGEMENTS We wish to thank Jane den Dunnen-Brigss, Jettie van der Wijk,Klaas Heeres and Miranda Nijenhuis (Gröningen) for theirtechnical assistance and Hans-Jürg Alder and his staff (NucleicAcid Facility, Kimmel Cancer Center, Jefferson Medical College,Philadelphia, PA) for DNA synthesis and sequencing. AntibodyE3 against K17 was a generous gift of Dr S. Trojanovsky(Moscow). This work was supported by grants from the JanKornelis de Cock-Stichting (to M.F.J.), the Dystrophic Epider-molysis Bullosa Research Associations (DEBRA) of the UK andUSA (to W.H.I.M.) and by the US Public Health Service,National Institutes of Health (grant PO1-AR38923 to J.U.). NOTE ADDED IN PROOF Since submission of this manuscript, we have learned that anothergroup has identified a PC-2 family which showed linkage to thetype II keratin cluster. These data were presented at the 5th JointClinical Genetics Meeting, Los Angeles, CA, February 1998, byDrs D. Ross McLeod and Gail E. Graham, Alberta Children’sHospital, Calgary, Alberta (G.E. Graham, personal communica-tion). REFERENCES 1.Quinlan, R.A., Hutchison, C.J. and Lane, E.B. (1994) Intermediate filaments.In Sheterline, P. (ed.), Protein Profiles . Academic Press, London.2.Fuchs, E. and Weber, K. (1994) Intermediate filaments: structure, dynamics,function and disease.  Annu. Rev. Biochem ., 63 , 345–382.3.McLean, W.H.I. and Lane, E.B. (1995) Intermediate filaments in disease. Curr. Opin. Cell Biol ., 7 , 118–125.4.Corden, L.D. and McLean, W.H.I. (1996) Human keratin diseases: hereditaryfragility of specific epithelial tissues.  Exp. Dermatol ., 5 , 297–307.5.Irvine, A.D., Corden, L.D., Swensson, O., Swensson, B., Moore, J.E., Frazer,D.G., Smith, F.J.D., Knowlton, R.G., Christophers, E. et al.  (1997) Mutationsin cornea-specific keratins K3 or K12 cause Meesmann’s corneal dystrophy.  Nature Genet  ., 16 , 184–187.6.Winter, H., Rogers, M.A., Langbein, L., Stevens, H.P., Leigh, I.M., Labreze,C., Roul, S., Taieb, A., Kreig, T. and Schweizer, J. (1997) Mutations in the haircortex keratin hHb6 cause the inherited hair disease monilethrix.  NatureGenet  ., 16 , 372–374.7.Winter, H., Rogers, M.A., Gebhardt, M., Wollina, U., Boxall, L., Chitayat, D.,Babul-Hirji, R., Stevens, H.P., Zlotogorski, A. and Schweizer, J. (1997) Anew mutation in the type II hair cortex keratin hHb1 involved in the inheritedhair disorder monilethrix.  Hum. Genet  ., 101 , 165–169.8.Ku, N.O., Wright, T.L., Terrault, N.A., Gish, R. and Omary, M.B. (1997)Mutation of human keratin 18 in association with cryptogenic cirrhosis.  J.Clin. Invest  ., 99 , 19–23.9.Stevens, H.P., Kelsell, D.P., Bryant, S.P., Bishop, D.T., Spurr, N.K.,Weissensbach, J., Marger, D., Marger, R.S. and Leigh, I.M. (1996) Linkage of an American pedigree with palmoplantar keratoderma and malignancy(palmoplantar ectodermal dysplasia type III) to 17q24. Literature survey andproposed updated classification of the keratodermas.  Arch. Dermatol ., 132 ,640–651.
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