Hum Genet (1992) 90:113-116 human ..

genetics �9 Springer-Verlag 1992

Mapping of a gene for epidermolytic palmoplantar keratoderma to the region of the acidic keratin gene cluster at 17q12-q21

Andr6 Reis 1, Wolfgang Kiister 2, Ralf Eckardt 1, and Karl Sperling 1

lInstitut ft~r Humangenetik, Freie Universit~it Berlin, Heubnerweg 6, W-1000 Berlin 19, Federal Republic of Germany 2 Universit~itshautklinik, W-3550 Marburg, Federal Republic of Germany

Received December 12, 1991

Summary. Epidermolytic palmoplantar keratoderma (EPPK) (V6rner-Unna-Thost) is an autosomal domi- nantly inherited skin disease of unknown etiology char- acterized by diffuse severe hyperkeratosis of the palms and soles and, histologically, by cellular degeneration. We have mapped a gene for EPPK to chromosome 17qll- q23, with linkage analysis using microsatellite DNA- polymorphisms, in a single large family of 7 generations. A maximum lod score of z = 6.66 was obtained with the probe D17S579 at a recombination fraction of 0 = 0.00. This locus maps to the same region as the type I (acidic) keratin gene cluster. Keratins, members of the inter- mediate filament family, the major proteins of the cyto- skeleton in epidermis, are differentially expressed in a tissue-specific manner. One acidic keratin, keratin 9 (KRT9), is expressed only in the terminally differen- tiated epidermis of palms and soles. The KRT9 gene has not yet been cloned; however, since the genes for most acidic keratins are clustered, it is highly probable that it too will map to this region. We therefore propose KRT9 as the candidate gene for EPPK.

Introduction

Epidermolytic palmoplantar keratoderma (EPPK) was first described by V0rner in 1901. It is an autosomal domi- nantly inherited disorder (McKusick 1990, no. 144200) that is clinically characterized by diffuse yellow kera- toses, sharply bordered by erythematous margins, over the entire surface of the palms and soles. Histologically, the disease presents the characteristic features of epider- molytic hyperkeratosis consisting of perinuclear vacuoli- zation of the keratinocytes and large irregularly shaped keratohyalin granules located in the granular layer of the epidermis.

A second, clinically identical palmoplantar kerato- derma, also with autosomal dominant inheritance, is

Correspondence to: A. Reis

known as palmoplantar keratoderma type Unna-Thost (McKusick 1990, no. 148400); Thost 1880; Unna 1883). The histopathology is believed to be non-specific and the disease has been termed the most common hereditary disorder with regard to the keratinization of the hands and feet (DerKaloustian and Kurban 1979). However, recent data obtained from a reappraisal of the kindred originally analysed by Thost (1880) has brought into question the idea of two separate genetic entities leading to diffuse palmoplantar keratoderma. Indeed, a descen- dant from this family exhibits all the features of EPPK, as described by V6rner, thus giving evidence for the identity of both conditions (Ktister and Becker 1992). Neither the underlying biochemical defect nor the loca- tion of the gene(s) is known.

Microsatellite DNA-polymorphisms, also called sim- ple sequence length polymorphisms (SSLPs; Hamada et al. 1982; Nordheim and Rich 1983; Tautz and Renz 1984), are ideal for linkage analyses of less common genetic dis- eases or when only a limited number of affected families is available for study (Reis 1991). They consist of tan- demly repeated simple sequence motifs, mainly dinucleo- tides; they are evenly distributed over the genome and can be easily detected by the polymerase chain reaction (PCR) (Litt and Luty 1989; Tautz 1989; Weber and May 1989; Zuliani and Hobbs 1990). Most microsatellite poly- morphisms are highly informative because of their large number of alleles. To date, more then 200 microsatellite DNA-polymorphisms with varying degrees of informativ- ity have been described, both in previously mapped gene loci and in anonymous DNA loci (A. Reis, unpublished). In this report, we have used such microsatellites for mapping the EPPK gene in one large kindred of 7 genera- tions.

Subjects and methods

Family investigation

A large kindred with EPPK from Westfalia (Northwest Germany) was ascertained by one of the authors (W.K.). The pedigree in-

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volves a family of 7 generations most individuals of the last 3 gen- erations are alive and cooperative, and several branches of the family live in different parts of Germany. The portion of the pedi- gree that is relevant for the linkage analysis is shown in Fig. 1. The diagnosis of EPPK was made after clinical examination and was verified by histopathological investigations. The ascertainment of the affected status of most probands relied solely on clinical ex- amination.

Microsatellite analysis

Blood samples (10 ml) were obtained from all consenting members of the kindred and DNA was prepared as described previously (Cooper et al. 1985). Microsatellite DNA-polymorphisms were amplified by PCR with Thermus aquaticus (Taq) DNA polymerase (Saiki et al. 1988) in an automated Thermal Cycler (Perkin-Elmer- Cetus). In this study, we analysed the following loci: D17S250 (Weber et al. 1990), D17S579 (Hall et al. 1992) and GH (Poly- meropoulos et al. 1990), which have been mapped via somatic cell hybrids to chromosome regions 17@1.2-@2, 17q12-q23 and 17@2-24, respectively.

Each reaction was performed in a 10 to 15-~tl volume contain- ing 5-10 pmol of each primer, 200 I-tM each dGTP, dCTP, dTTP, dATP, 10raM TRIS-HC1, 50mM KCI, 2.5mM MgC12, 0.01% (w/v) gelatin and 0.5 U AmpliTaq DNA polymerase (Perkin-EImer- Cetus) and overlaid with mineral oil. One of the primers was previ- ously end-labelled for 30 min at 37~ in a reaction containing 30 pmol primer, 20gCi gamma 32p-ATP, (3000mCi/mol) and 5 U polynucleotide kinase (Pharmacia) under the conditions recom- mended by the supplier. After an initial denaturation step of 7 rain at 94~ amplifications were performed for 27 cycles as follows: I rain at 94~ 1 rain at 55~ and 1.5 min at 72~ A final incuba- tion for 10 rain at 72~ was made to ensure complete elongation of the products. Subsequently, 5-[al aliquots were electrophoresed on 8% denaturing polyacrytamide gels. The gels were then fixed and dried, and autoradiograms exposed overnight at 70~ A repre- sentative example is shown in Fig. 1.

Linkage analysis

Two-point linkage analysis was performed using the MLINK com- puter program, version 3.5 (Ott 1976). Autosomal dominant in- heritance with full penetrance was assumed. Since no recombinants were observed between EPPK and D17S250 or D17S579, no multi- point analysis could be performed.

Results

A tota l of 29 ind iv idua ls f rom the k ind red could be ana- lysed, 9 of w h o m were classif ied as affected. Bo th loci D17S250 and D17S579 co- segrega ted with E P P K in the family ana lysed and no r ecombinan t s were obse rved . Al l a f fec ted indiv iduals had one c o m m o n al lele at D17S579, whereas none of the non-a f fec ted family m e m b e r s ex- h ib i ted this al lele (Fig. 1). In con t ras t to D17S579, which was fully in fo rmat ive , D17S250 was only in format ive in one b ranch of the k indred . A m a x i m u m lod score of z = 6.66 was o b t a i n e d for D17S579 at a r e c o m b i n a t i o n frac- t ion of 0 = 0.00, and of z = 3.18 at 0 = 0.00 for D17S250.

Discussion

Localization of a gene for EPPK

W e have m a p p e d a gene for ep ide rmo ly t i c p a l m o p l a n t a r k e r a t o d e r m a ( E P P K ) with l inkage analysis to ch romo-

Fig. 1. Pedigree of the portion of the kindred with EPPK relevant for the linkage analysis, and the corresponding autoradiogram of the microsatellite at D17S579. Filled symbols indicate affected pro- bands, open symbols non-affected probands. Each allele of the microsatellite is characterized by one intense band and a number of spurious bands. All affected probands share the upper allele but none of the non-affected descendants have this allele

some 17q12-q21 in one single family of 7 genera t ions using microsa te l l i t e D N A - p o l y m o r p h i s m s . No r ecombi - na t ions were obse rved , ne i the r be tween D17S250 nor D17S579 and E P P K in this family. D17S250 was only par t ia l ly in fo rmat ive , desp i t e its high p o l y m o r p h i s m in- f o rma t ion con ten t value. The m a x i m u m lod-score ob- t a ined with D17S579 was z = 6.66 at 0 = 0.00. Al l indi- v iduals a f fec ted had one al lele at this locus in c o m m o n , whereas none of the non-a f fec ted family m e m b e r s ex- h ib i ted this al lele. This excludes the poss ibi l i ty of r ecom- b ina t ion b e t w e e n E P P K and this locus in the gene ra t ion for which phases could not be ident i f ied with cer ta in ty because of d e c e a s e d p robands .

Severa l r e combinan t s were o b s e r v e d with the micro- sa te l l i t e D N A - p o l y m o r p h i s m s at the g r o w t h - h o r m o n e ( G H ) locus, loca ted distal to D17S579 at 17q22-24. Since we have not o b s e r v e d any r ecombinan t s be tween the two cen t romer i c loci and E P P K in this family, the impli- ca t ion is that E P P K is loca ted p rox ima l ly within the re- g ion 17q11.2-q23, de f ined by the c o m b i n e d loca l iza t ion data . Recen t work on genet ic maps f rom this reg ion by Hal l and co -worke r s (1992) p laces D17S579 approxi - ma te ly 6 cM t e lomer i c to D17S250 and b e t w e e n H E R 2 and GIP .

Candidate gene for EPPK

The family of type I (acidic) ke ra t in ( K R T ) genes has been m a p p e d by somat ic cell hybr id analysis and in situ hybr id iza t ion techniques to c h r o m o s o m e 17q11-21 (Les- sin et al. 1988; R o m a n o et al. 1988; R o s e n b e r g et al. 1988). This gene family includes KRT10 , which is the ma jo r type I ke ra t in of s t ra t i f ied ep i the l ia (S to ler et al. 1988; Lessin et al. 1988), KRT14 , which is found in basal epithelial cells (Nelson and Sun 1983) and KRT16, which

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is found in hyperproliferative suprabasal cells (Weiss et al. 1984). Depending on the stage of differentiation, all these proteins are present in stratified human epidermis, including palmar and plantar epidermis. Palms and soles exhibit one additional keratin not found in most other epi- dermis, viz. KRT9 (Moll et al. 1982; Knapp et al. 1986), which is only present in the terminally differentiated cells of the stratum spinosum and stratum corneum (Fuchs and Green 1980; Moll et al. 1987). Acidic keratins form heterodimers with basic or type II keratins to produce the intermediate filaments that are the major component of the cytoskeleton in epidermal cells. Most of the type II keratin genes have been mapped to chromosome 12q12 (Lessing et al. 1988; Rosenberg et al. 1991).

The localization of EPPK to the same chromosomal region as the acidic keratin gene cluster is highly sugges- tive of the involvement of a keratin gene in the etiology of this disease. Since one keratin gene, KRT9, is known to be strongly expressed only in palmar and plantar epi- dermal cells, this gene is a natural candidate gene for EPPK. Furthermore, changes in structural proteins such as the keratins, which always function as heterodimers, could explain the dominant phenotype of this disease: even quantitative alterations of only one member of the dimer will affect the dimer as a whole. The gene for KRT9 has not yet been cloned; however, it has been shown, by in vitro translation analysis and subsequent detection with specific antibodies, that it is encoded by a discrete mRNA and that it is not a product of proteolysis or alternate splicing of other keratin genes (Knapp et al. 1986). This data, together with the findings described here, lead us to propose KRT9 as a candidate gene for EPPK. We are currently pursuing the cloning of KRT9, in order to test this hypothesis.

Heterogeneity

The localization of a gene for EPK in one single family precludes any problems resulting from possible genetic heterogeneity of the disease, since we can assume that the same mutant allele is segregating in this kindred. We have also been able to analyse the family originally de- scribed by Thost (1880), although only five descendants, four of whom were affected, were available for the anal- ysis. The family was informative and no recombinants were observed (data not shown). These results are there- fore fully compatible with our chromosomal localization of EPPK. These findings together with those of Kiister and Becker (1992) indicate that much of the effort in the sub-classification of EPPK into different disease entities or sub-types has probably been in vain. We propose now to sub-classify EPPK primarily only into those forms that are linked to the acidic keratin gene cluster and those, if any, that are not linked.

It is of interest that most patients with EPPK come from families in which the disease has occurred for many generations, as in the family described here. Thus, it seems reasonable to assume that the number of patients with EPPK caused by new mutations will be small, and that most cases of EPPK, at least within one geographi- cal area, have arisen from the same mutation.

Other related conditions

Since EPPK is a keratinization disease of palms and soles, we suggest that other disorders of the skin involving ker- tinization processes, for example the ichthyoses, are also caused by mutations in the keratin genes. Linkage to either keratin gene cluster on chromosomes 17q11-21 or 12q12 should further verify this hypothesis. This approach has recently led to the elucidation of the basic defect causing epidermolysis bullosa simplex type Dowling- Meara. Mutations in a basal keratin gene, KRT5, and in an acidic keratin, KRT14 have been described in different patients with this disease (Bonifas et al. 1991; Coulombe et al. 1991).

EPPK has been described in some families in con- junction with other diseases. Blanchet-Bardon et al. (1987) have reported a large kindred in which EPPK oc- curred in association with breast or ovarian cancer, or both. Recently, King's group has reported the localization of an early-onset familial breast cancer gene (BRCA1) to the same chromosomal region (Hall et al. 1990). Their original findings were made using D17S74, but their most recent results have pointed to a more proximal lo- cation, D17S579, being the closest marker at 0= 0.00 (z -- 9.12) (Hall et al. 1992). It therefore seems probable that both genes were mutated in this family and co-segre- gated, indicating close linkage of BRCA1 and EPPK. A major mutation affecting both genes could also be pos- sible if both genes were in close physical proximity. This is also indirect evidence that, in the family that we report here, the same gene might be affected as in the one re- ported by Blanchet-Bardon et al. (1987) further substan- tiating the proposal of genetic homogeneity in EPPK.

Rabbiosi et al. (1980) have described a family show- ing close association between EPPK and Charcot-Marie- Tooth disease type II (CMT2) with motor and sensory neuropathy (McKusick 1990, no. 118210). In their re- port, all members of the family with EPPK also had clear signs of motor and sensory neuropathy with the excep- tion of the younger generation, in which the probands were probably too young clearly to manifest CMT2. The histological findings of EPPK described by Rabbiosi et al. (1980) are typical for EPPK and do not differ from those in the kindred described here. Given that CMT2 is not allelic to either the CMT1A locus on chromosome 17p or the CMT1B locus on chromosome lq21-q25 (Hentati et al. 1992), and assuming genetic homogeneity in EPPK, our results suggest the localization of at least one gene for CMT2 to proximal 17q.

Acknowledgements. We are indebted to all members of the family with EPPK for their cooperation in this study. We thank Ladislaus Ladanyi for technical assistance and Martin Digweed for a critical review of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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