Evidence Against Keratin Gene Mutations in a Family with Ichthyosis Hystrix Curth-Macklin

RAPID COMMUNICATION Evidence Against Keratin Gene Mutations in a Family with Ichthyosis Hystrix eurth-Macklin

Jeannette M. Bonifas, John W. Bare, Marisa A. Chen, Annamari Ranki,* Kirsti-Maria Neimi,* and Ervin H. Epstein, Jr. Departments of Dermatology University of Cali fornia School of Medicine, San Francisco, California, U.S.A.; and 'Helsinki University Central Hospital, Helsinki, Finland

Ichthyosis hystrix Curth-Macklin is a rare autosomal domi might underlie this disease. This analysis excluded the kera nant disease characterized clinically by hyperkeratosis and tin gene loci as the sites for the disease-causing mutation in ultrastructurally by disruption of the keratin intermediate one affected kindred. Key words: linkage analysis/epidermal filament network of suprabasal keratinocytes. W e have used disease/ genodermatoses/ epidermolytic hyperkeratosis. ] In linkage analysis to test whether a keratin gene mutation vest DermatoI101:890-891, 1993

lthough many cutaneous disorders have been termed MATERIALS AND METHODS "disorders of keratinization," only very recently have DNA was isolated from peripheral blood leukocytes. After informed consent phenotypic abnormalities resulting from keratin ge ne 30-60 ml blood was drawn from each patient. After anticoagulation with abnormalities been elucidated. Specifically, there now ethylenediamine tetraacetic acid. the lymphocytes were separated with Fi is substantial evidence that mutations of ge nes encod coli density centrifugation (Fi co ll -Paque, Pharmacia, Uppsala) and washed. Aing keratins expressed in basal keratinocytes underlie epidermolysis and high - molecular-weight DNA was iso lated. The cells were first lysed in mM , bullosa simplext [1 - 6] and that mutations of genes encoding kera Tris-HCI buffer (pH 7.6) containing 1 % Triton-X-I00. 5 MgCl2 and tins expressed in suprabasal keratinocytes underlie generalized epi 320 mM sucrose, washed, and digested in ethylenediamine tetraacetic acid dermolytic hyperkeratosis (EHK) (bullous congenital ichthyosi NaCI so lution with 1% sodiu mdodecyl sulfide and proteinase K (0.1 mg! form erythroderma) [7 -13]. ml; Merck. Darmstadt. Germany). The DNA was extracted once each with One very rare disorder of keratinization is ichthyosis hystrix TE-phenol. phenol : chloroform, and chloroform, and then precipitated with ethanol and 0.3M NaAc. The precipitate was was hed with 70% eth Curth-Macklin (IHCM). During the past four decades two families anol. dissolved in TE-buffer. and stored at - 20·C until used. and some isolated patients with this disease have been described Polymorphic loci analyzed are known to be close to the type II keratin [14-20]. In the former, inheritance was compatible with an autoso gene cluster on chromosome 12 and to the type I keratin gene cluster on mal dominant trait. The typical clinical abnormality is hyperkerato chromosome 17 (Chen, MA et ai, manuscript in preparation). Specifically, sis of somewhat variable extent (localized to generalized) and sever Mspl-digested genomic DNA was subjected to Southern analysis at D 12S1 4 ity', both within one family and at different times within one by hybridization with pEFD33 .2 (21). at D12S17 by hybridization with individual. Abnormalities of the middle and upper spinous kerati pYNH15 [22]. and at KRT 5 by hybridization with a cDNA probe (Chen nocytes detectable by electron microscopy arc characteristic, with MA. Epstein E. Jr .• manuscript in preparation). Hinfl-digested genomic' thick perinuclear shell s comprised of intermediate fil aments fl anked DNA was subjected to Southern analysis at D17S74 by hybridization with pCMM86 [23]. The microsatellite repeat polymorphism at D17S579 was centrally by a region rich in ribosomes and peripherally by normal analyzed by gel e1eccrophoresis of products of polymerase chain reaction intermediate fil ament bundles terminating on desmosomes. This using 32P-label ed primers [24 -26]. Two-point linkage analysis was calcu appearance is found in both clinically normal and affected skin lated with LIPED [27). [1 6-20] . These histologic findings resemble but arc distinct from those of EHK; tonofilament clumping typical in EHK is lacking in RESULTS IHCM. C linical findings in this kindred (IHCM-Ko) of hyperkeratosis of We have studied DNA from one of the two known kindreds, early onset and localization to the ankles, elbows, and knees have IHCM-Ko [16] . Patients in this family have thick, furrowed-ap been described [1 6]. When inheritance of the abnormal phenotype pearing hyperkeratosis localized over joints. W e report here evi was compared with inheritance of polymorphic loci ncar the type II dence against keratin gene mutations being the inherited defect keratin gene cluster on chromosome 12q and the type I keratin gene underl ying this disease. cluster on chromosome 17q, recombinants were noted with each (Fig 1, Table I) . This provides strong evidence against keratin genes being the site of the mutation underlying the disease in this kindred. DlSCUSSION Manuscript received July 6, 1993; accepted for publication August 25, The ultrastructural changes in IHCM do differ from those of epi 1993. dermolytic hyperkeratosis. However, in both diseases there are ab Reprint requests to: Dr. Ervin Epstein,Jr., Building 100, Room 269, San Francisco General Hospital, 1001 Potrero Street, San Francisco. CA 94110. normalities of the keratin intermediate filament network of the cells Abbreviations: EHK. epidermolytic hyperkeratosis; IHCM. ichthyosis of the upper layers of the epidermis. In EHK the tonofilaments form hystrix Curth-Macklin. tight bundles or balls with sharp margins. In contrast, the tonofila t Hoyheim B. Gedde-Dahl T Jr. Berg E. Oiaisen B: Linkage studies in ments in IHCM seem to be unabl e to form regular. parallel tonofi epidermolysis bull osa simplex (abstr). J IrlVes! Dem!a!oI 98:397. 1992. lament bundles and instead fill so me of the upper keratinocytes

2 3. Vassar R, Coulombe PA, Degenstein L, Albers K, Fuchs E: Mutant keratin ex pression in transgenic mice causes marked abnormalities resembling a human genetic ski n disease. Cell 64:365 - 380, 1991 24 D12514 4. Ryynanen M, Knowlton R, Uitto J: Mapping of epidermolys is bullosa simplex 13 0175579 mutation to chromosome 12. Am] Hum Gellet 49:978-984,1991 5. Lane EB, Rugg EL, Navs.ria H, Leigh 1M, Heagerry AHM, Ishida-Yamamoto A, Eady RA]: Mutation in the conserved helix termination peptide of keratin 5 in II 6 hereditary skin blistering. Nature 356:244-246,1992 6. Epstein EH Jr: Molecular genetics ofep idermolysis bullosa. ScietJce 256:799 -804, 1992 7. Compton]G, DiGiovanna]], Santucci SK, Kearns RS, Amos CI, Abangan DL, Korge BP, McBnde OW, Steme.rt PM, Bale S]: Linkage of epidermolyric hyperkeratoSIS to the rype II keratin gene cl uster on chromosome 12q. Nature Gellct 1:301 -305, 1993 III 4 8. Bonifas JM, Bare ]W, Chen MA, Lee MK, Slater CA, Goldsmith LA, Epstein EH 12 24 12 Jr: Lmkage of the ep.dermolytlc hyperkeratosis phenorype and the region of the rype II keratin gene cluster On chromosome 12.) [liVest DermatoI 99:524- 35 23 12 12 527,1992 9. Pulkkinen L, C hri s ti an ~ AM, Knowlton RG, Uitto]: Epidermolyric hyperkera Figure 1. Genotypes of members of IHeM-Ko kindred for polymorphic tOSIS (bullous congemtal .chthyoSlform erythroderma).] elill [tlvest 91:357- loci near the keratin gene clusters on chromosome 12 (D12S14) and chro 361,1993 mOSOITle 17 (D17S579). Solid symbols, affected persons; oprll symbols, unaf 10. Rothnagcl JA, Dominey AM, Dempsey LD, Longley MA, Greenhalgh DA, fected persons; squares, male, circles, female. Gagne TA, Huber M, Frenk E, Hohl D, Roop DR: Mutations in the rod dom:uns of keratllls 1 and 10 in epidermolytic hyperkeratosis. Sciwu 257:1128- 1 130, 1992 II. Che'~ g ], Syder AJ, Yu QC, Letai A, Paller AS, Fuchs E: The genetic basis of Table I. Pairwise Lod Scores Between IHCM-Ko and ep.dermolyttc hyperkeratosis: a disorder of differentiation-specific epidermal Chromosome 12q and 17q Loci" keratin genes. Cell 70:81 1-819, 1992 12. C hi p~v CC, Korge BP, Markova N, Bale S], DiGiovanna ]], Compton ]G, Lod score at 0 of Stemert PM: A leucllle-proline mutation in the HI subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 70:821-828, 1992 0.001 0.01 0.05 0.10 13. Fuchs E, Esteves RA, Coulombe PA: Transgenic mice expressing a mutant keratin 10 gene hkely generic basis for cpidermolytic hyperkeratosis. Proc Nat! Acad Sci 0 12S14 -7.55 -4.55 -2.46 -1.59 USA 89:6906-6910,1992 012S17 -3.29 -2.29 -1.52 -1.10 14. Olle l:dorff-C~rth H , Macklin MT: The genetic basis of various types of ichthyo KRT 5 -3.33 -2.30 -1.46 -1.03 SIS III a fanllly group. A m] Humatl Get,ct 6:371-382, 1954 15. Ollendorff-Cunh H, Allen FH J r. , Schnyder UW, Anton-Lamprecht I: Follow 017S579 -6.59 -3.62 -1.65 -0.91 up of a fanuly group suffenng from ichthyosis hysrrix rype Curth-Macklin. 0 17S74 -4.41 -2.43 -1.09 -0.57 HlunatlgCllctik 17:37 -48, 1972 16. Niemi K-M, Virtanen I, Kanerva L, Muttil. inen M: Altered keratin expression in • Paternal and maternal segregarion are combined, and autosomal dominanr inheri . ichthyosis hystrix Curth-Macklin. Arch Dermatol R es 282:227 - 233, 1990 tance with full penetrance and no sporadic Cases has been assumed. 17. Plllkus H: Nagao S: A case of biphasic ichthyosiform dermatosis: light and elec tron mIcroscopic study. A rch Klill Exp Derm 237:737 - 748, 1970 18. Anton-Lamprecht I, Kern B, Goerz G, Marghescu S: Perinuclear shell fo rmation III uncommon ichthyoses. ] Cllla tl Patho/8:447 - 448, 1981 with a haphazardly arrayed filament network. Binucleated ceIls are 19. Kanerva L, Karvonen J, Oikarinen A, Lauharanta J, Ruokonen A, Niemi K-M: Ichthyosis hystrix (Curth-Macklin). Arch Dermatol 120:12 18-1223 present in both human diseases as well as in transgenic mice carrying 1984 ' genes encoding mutant keratin 10 [13]. Hence the lack oflinkage to 20. Anton-Lamprecht I, Curth HO, Schnyder UW: Zur ultrastruktur hereditarer keratin genes in this IHCM kindred is an interesting contrast to the Verhornungsstorungen. Arch Derm Forsch 246:77-91, 1973 linkage and sequence analyses in EHK, which identify underlying 21. Nakamura Y, Fujimoto E, Marrin C, O'Connell P, Leppert M, Lalouel JM, Willte R: Isolation and mapping of a polymorphic DNA sequence pEFD33.2 mutations of keratins 1 and 1 O. We conclude that abnormalities of on chromosome 12 (DI2S14). Nucleic Acids Res 16:778, 1988 genes encoding proteins other than keratins may disrupt the keratin 22. N aka~ ura Y, Ballard L, O'Connell P, Leppert M, Lathrop GM, Lalouel JM, intermediate filament network and may be associated with binu Willte R: Isolation and mapping of a polymorphic DNA sequence p YNH15 on cleated keratinocytes. For example, the finding of disruption of the chromosome 12q (DI2S17). Nucleic Acids Res 16:779.1988 23. Nakamura Y, Martin C, Myers R, Ba.llard L, Leppert M, O'Connell P, Lathrop intermediate filament network following transfection of DNA en GM, Lalouel JM, White R: Isolation and mapping of a polymorphic DNA coding a mutant desmoplakin [28] is compatible with that conclu sequence (PCMM86) on chromosome 17q [DI7S74]. Nucleic Acids Res sion. 16:5223, 1988 24. Weber JL, May PE: Abundant class of human DNA polymorphisms which can be ryped uSlllg the polymerase chain reaction. A m] Hum Gwet 44:388-396 1~9 ' This work was supported in part by NIH grallt AR 41120 alld by a grant from 25. 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