Keratin 5 polymorphism in PPK patients 
C linica/ and laboratory study 
KERATIN 5 ASSOCIATED RESTRICTION FRAGMENT 
LENGTH POLYMORPHISM IN PATIENTS WITH 
PALMOPLANTAR KERATODERMA OF UNNA-THOST 
TYPE 
S.Tomic, R.Komel, A.Kansky and M.Blumenberg 
ABSTRACT 
Hereditary palmoplantar keratodermas of the Unna-Thost type (HPPK-UT) are relatively frequent among the Slovenian and 
Croatian population. Members of two Slovenian families (33 persons altogether) were investigated for RFLP (restriction 
fragment length polymorphism). RFLP of the patients DNA was studied by applying the following endonucleases: Hind III, 
Taq I, EcoRI, Sac I, BamHl and Msp I. Only digestion with Msp I produced four polymorphic fragments which were identified 
asa, b, c and d alleles ofkeratin 5. 14 out of 15 affected persons expressed the d allele in a heterozygous or homozygous way. 
In a number of unaffected members the d allele was also present. 
KEYWORDS 
Keratin 5, restrictionfragment length polymorphism, pa/moplantar keratoderma , Unna-Thost type, Msp I 
INTRODUCTION 
Hereditary palmoplantar keratodermas (HPPK) are 
disorders of keratinization characterized by a thickened 
homy layer on palms and so les. V arious types of HPPK are 
distinguishable by clinical symptoms , associated 
abnormalities and the mode of inheritance. HPPK of the 
Unna-Thost (UT) type is the most frequent, its prevalence is 
1:40.000 in Northem Ireland (1), 1:12.000 in Slovenia (2) 
and 1:25.000 in Croatia (3). In Sweden at Westerbotten 
county a prevalence of 1:300 among adolescents (4) and at 
Norbotten county of 1:200 among school children were 
registered (5). The cause of HPPK - UT is not known, 
however dominant inheritance and circumscri bed distribu tion 
allow the possibility that this disease is due to a mutation in 
a structural protein specialll y expressed in palmar and plan tar 
epidermis. Likely candidates for such proteins are keratins. 
Keratins are inte1mediate filament proteins which constitute 
the cytoskeleton of all epithelial cells. Over 30 such proteins 
are known and each is coded by a separate gene. They can be 
subdivided into two distinct classes on the basis of their 
migration in two dimensional electrophoresis. theirexpression 
in different cell types and of their sequence homology (6). 
Type I keratins (KlO - K19 are smaller (40-56.5 kD) and 
This work was supported by the V .S . Joint Board project HSS JF 915 "Genetic Studies on Hereditary Disorders of Keratinization " which is 
gratefully acknoYi-"ledged. 
114 acta dermatovenerologica A.P.A . Vol 1, 92 , No 4 
Keratin 5 polymorphism in PPK patients 
relatively acidic (pI 4.5-5.5), whereas type II keratins (Kl -
K9) are larger (53-68 kD) and more basic (pI 5.5-7.5). 
Usually keratins are expressed as specific pairs consisting of 
one type I and one type II polypeptide, both of which are 
essential for filament formati on (7 ,8) and as such characterize 
biochemically the type of epithelial differentiation. Por 
example KS and K 14 are synthesized in the basal cell layer 
of ali stratified squamous epithelia, while in the course of 
stratification differentiating epidermal cells express Kl and 
K 10. Suprabasal maturing cells ofnonkeratinizing squamous 
epithelia express K4 and K 13, while in a number of epidermal 
skin diseases K6 and K16 are expressed suprabasally (9). 
Expression ofkeratins is often much greater in differentiating 
cells than in basal cells (10). 
Elucidating ofkeratin functions has been hampered by Jack 
of known genetic disease involving an identified keratin 
mutation. During the last few years however important new 
data concerning the genetics of keratin were accumulated. 
Hereditary epidermolysis buli osa simplex (EBS) arises from 
basal celi cytolysis. Coulombe et al. (11) demonstrated that 
a perturbation of basal celi keratin filament network was 
responsible for the clinical manifestations of EBS. In two 
patients with EBS of Dowling-Meara type a point mutation 
in thecritical region ofthe K14 gene was observed: Arg 125 
to Cys mutation. The same genetic defect in transfected 
keratinocytes resulted in a disruptedkeratinnetworkfo1mation 
and in a perturbarted filament assembly. In a family with 
EBS ofKoebner type Bonifas et al. (12) were able to map the 
defect to chromosome 17 by !od score calculation and to 
identify the mu tati on in the base pair 3542 of the responsible 
gene. Lane et al. (13) described in a family with EBS of 
Dowling-Meara type a Glu to Gly mutation in the helix-
termination peptide of keratin 5. It is worth to mention that 
Vassar et al. (14) by inse1ting a truncated Kl4 protein 
(missing 135 amino acid residues at the C-terminal) into 
mice succeeded to produce epide1mal blistering in transgenic 
offspring which was similar to EBS lesions. The 
micromorphologic investigation revealed a disruption of 
keratin network assembly, presence of clumped keratin and 
cytolysis of basal cells. Complementary in vitro filament 
assembly studies on bacterially produced human KS, Kl4, 
and the truncated mutant K 14 showed that addition of as little 
as 1 % ofthe mutant K14 to the assembly mixture caused a 
detectable alteration in filament formation (14). Purther 
transgenic studies confirmed the dominant behavior of 
mutations such as the above mutation in K14, and provided 
strong evidences as to which human skin diseases might be 
candidates for natura! mutations not only in K14 but also in 
other genes such as KS. More recently another hereditary 
epidermal disorder has been traced to mutations in keratin 
genes: epide1molytic hyperkeratosis (EH) which is due to 
mutationsintheKl andKlOkeratingenes( 15, 16, 17, 18). 
acta dermatovenerologica A.P A. Vol 1, 92 . No 4 
Therefore we examined the possibilitiy that HPPK-UT 
mutation maps in the type II keratin gene cl uster, by examining 
genetic linkage betweenHPPK-UT and a pol ymorphic mar ker 
in the KS keratin gene. 
The human KS keratin gene encodes a 58 kD protein which 
belongs to the basic Type II keratin family. The protein is 
polymorphic and at least two allelic variants were detected 
(19,20). The gene itself is approximately 5 kb long, contains 
nine exons and is located on the chromosome 12 (21,22). In 
the present study we are reporting some evidence about 
polymorphism within the coding region of the gene itself. 
MATERIALS AND METHODS 
Blood samples were taken from members of two Slovenian 
families affected by HPPK-UT as well as from non-affected 
and nonrelated controls. Eight patients and nine non-affected 
relatives belonged to family SO from Eastern Slovenia (Celje 
area). Another seven patients and nine non-affected persons 
were members of family PA from Westem Slovenia 
(Ajdovščina area). 
High molecular weight DNA was prepared from nuclei of 
leukocytes. Samples of the whole blood were first lysed by 
adding an equal volume of solution containing 0.32 M 
sucrose, 10 mM Tris-HCl (pH 7.4), 5 mM MgCl
2 
and 1 % 
Tri ton X-100. The nuclei were then collected by centrifugation 
and theDNA was purified by incubating ovemight at 37°C in 
a buffer solution of 10 mM Tris-HCI, 400 mM NaCl and 
2 mMEDT A (pH 8.2) containing 1 % sodi um dodecyl sulfate 
(SDS) and 0.15 mg/ml Proteinase K. Proteins were salted out 
by 6M NaCl and DNA was precipitated from the supernatant 
by adding two volumes of ice-cold absolute ethanol (23). 
Digestions of the DNA with restriction endonucleases 
such as Hind III, Taq I, Eco RI, Sac I, Bam HI and Msp I were 
canied out observing the conditions recommended by the 
manufacturer (Gibco BRL). The digested DNAs were 
fractioned by electrophoresis in 0.8 % agarose gel. 
After electrophoresis the digested DNA was denatured in 
the gel by submersing in 0.4MNaOH and blotted onto nylon 
membranes (NEN DuPont) by capillary transfer (24). PKA
1 
probe containing a 1.68 bp DNA fragment of the KS gene 
(PKA
1
-bprobe) waslabelled withalpha/32P/-dCTPto aspecific 
activity of3x 108 cpm/µg by random primer extensionmethod 
(25) using the Amersham Multiprime labelling kit. 
Membranes with restricted and denatured DNA fragments 
were hybridized to radieJactively labelled PKA 1-b probe in a 
standard hybridization solution containing 50 % formamide 
115 
Keratin 5 polymorphism in PPK patients 
Figure 1: 
Southern blots of restricted DNAs as individualized by 
autoradiography. Familly SO. 
(Amersham Hybridization Protocols) at 42°C for 48 hours. 
After hybridization the membranes were washed once in 
2xSSC/0.1 % SDS for 5 minutes at room temperature, and a 
second tirne in the same solution at 65°C for 15 minutes. 
Visualization was performed by autoradiography using a 
X-ray film (X-Omat, Kodak), after two and seven days 
exposure times. 
RESULTS 
In order to search for KS-gene polymorphisms first six 
DNA samples from unaffected unrelated individuals were 
Figure 2: 
Southern blots of restricted DNAs as visualized by 
autoradiography. F amilly F A. 
116 
digested with six different restriction endonucleases and 
hybridized to PKA 1 probe. Five of the enzymes gave non-
polymorphic band pattems, but only digestion with Msp I 
produced polymorphic fragments of which the total number 
was four and with the lengths of 8.7, 7.2, 6.4 and 3.2 kb 
respectively. The fragments were assigned asa, b, c and d 
alleles (Figures 1 and 2). 
Two families with hereditary PKK were then screened for 
the presence of Msp I RFLP. The genotypes of both the 
families are presented. In the family SO (Eastem Slovenia) 
the eight affectedmembers displayed b/d and a/dheterozigous 
or d/d homozigous genotype (Fig 3). In the farnily FA 
(Westem Slovenia), six out of seven affected members 
expressed the d allele. Details are presented in Figure 4. 
DISCUSSION 
It is difficult to find out exactl y who was the first to describe 
the polymorphism of keratin molecules. It seems however 
that the introduction of sodium dodecyl sulfate polyacrylarnide 
gel electrophoresis as well as of modem chromatographic 
methods in the protein biochemistry were helpful in achieving 
essential pro gress in this direction. W ild and Mische described 
in 1986 the occurrence of keratins K4a and K4b as well as 
K5a and K5b as conspicuous doublets in the stratified 
epithelium lining the upperdigestive tractin some individuals, 
while in others only one of these variants was present. They 
assumed that a polymorphism ofthe respective keratin genes 
was responsible forthis heterogeneity (26). Later on the same 
authors made a similar observation analyzing the keratins of 
human epidermis by SDS PAGE and immunoblotting: basic 
keratins Kla, Klb, K5a and K5b, and acidic keratins KlOa 
and KlOb appeared either as doublets or as one or the other 
variant. In rare cases other acidic keratins tentatively 
designated as KlOd and KlOc were also observed (20). In 
1992 Korge et al. (27) confirmed the existence of the above 
mentioned four variants of Kl0, which represent four 
differently sized alleles. In order to answer the question 
whether these KlO variants are due to multiple genes or 
different alleles within the human population or are caused 
by different posttranslational RNA processing, they have 
looked for the nature of the polymorphism by analyzing 
respective gene sequences. Their investigation showed that 
the polymorphism was located in the V2 subdomain of the 
K 10 molecule. According to their experience the size 
polymorphism of the KlO gene is inherited as a normal 
Mendelian trait, and the differently sized products obtained 
by PCR amplification most likely represent different alleles 
of a single-copy gene per haploid genome. 
In our investigation we have looked for the possible 
existence of restrictionfragment length polymorphisms within 
acta dermatovenerologica A.P.A. Vol 1, 92, No 4 
Keratin 5 polymorphism in PPK patients 
bd 
ab ~b 
Figure 3: 
Geneological tree and inheritance of the KS po/ymorphic a/leles infamily SO. 
Figure 4: 
Geneologica/ tree and inheritance of the KS polymorphic alleles infamily FA. 
the coding region of the K5 gene.Results obtained in this 
study indicate thatK5 is also present, like KlO, infour allelic 
modifications. It is interesting to mention that allele d was 
found in almost all persons affected with HPPK type Unna-
Thost (in 14 out of 15 patients). As this allele was present also 
in certain unaffected individuals it can be assumed that the 
respective gene is not characteristic for HPPK-UT. 
Furthermore, in three individuals, 24, 27 and 31 of farni! y 
F A, the disease does not segregate with the K5 gene alleles. 
We concl ude from these preliminary results that the mu tati on 
causing HPPK-UT is probably not linked with the basic 
keratin genes on chromosome 12. 
Further investigation, in order to confirm the above results 
and to find possi bl y the informative pol ymorphisms associated 
with pathological phenotype (s), has to include much greater 
number of hereditary PKK cases. To define precisely the 
nature of the polymorphisms found in this investigation PCR 
amplification and sequence analyses of the DNAs should be 
performed. And finally, extensive analyses such as cloning 
and sequencing of keratin cDNAs, such as K5 and Kl4 
cDNAs from skin mRNAs, in a number of patients will be 
needed before the etiology ofthe disease is fully understood. 
REFERENCES 
l .Ebling F .J .G., Marks R., Rook A.: Disorders of Keratinization. 
In: Textbook ofDermatology (Rook A., Wilkinson D.S., Eibling 
F.J., ChampionR.H., Burton J.C., eds.), Blackwell, Oxford 1986: 
pp. 1452. 
2. Kansky A., Arzenšek J., Rode M., Strojan J .: Keratodermia 
palmoplantaris of the Unna-Thost type in Slovenia. Acta Derm. 
acta dermatovenerologica A.P.A. Vol 1, 92, No 4 
Venereol. (Stockholm) 1984; 140-143. 
3. Stanimirovic A., Kansky A.: Palmoplantar keratoderrna type 
Unna-Thost in Croatia. Acta Derm. lug. 1991; 18: 67-72. 
4. Larsson P., Liden S.: Prevalence of skin diseases among 
adolescents 12-16yearsof age. ActaDerm. Venereol. (Stockholm) 
1966; 60: 415-423. 
117 
Keratin 5 polymorphism in PPK patients 
5. Bergstroem C.: Keratodermia palmaris et plantaris. Nord.Med. 
1967; 78: 155-156. 
6. Moll R., Franke W.W., Schiller D.L., Geiger B., Krepler R.: 
The catalog of human cytokeratins: patterns of expression in 
normal epithelia, tumors and cultured cell 1982; 31: 11-34. 
7. Hatzfeld M., Franke W.W .: Pair formation and promiscuity of 
cytokeratins: formation in vitro of heterotrophic complexes and 
intermediate-sized filaments by homologous and heterologous 
recombination of purified polypeptides. J.Cell Biol. 1985; 101: 
1826-1841. 
8.Eichner R., Sun T. -T., Aebi U.: Theroleofkeratin subfamilies 
andkeratinpairsintheformationofhumanepidermalintermediate 
filaments. J . Cell Biol. 1986; 102:1767-1777. 
9. SunT. - T.,EichnerR. , Schermer A., Cooper D., NelsonD.W., 
Weiss R.A.: Classification, expression and possible mechanism 
of evolution of mamalian keratin of unifying model. In: The 
Cancer Celi (Levine A.J., Topp W.C., Van de Wonde G.F., 
Watson J.D., eds.) Cold Spring Harbor Laboratory, New York 
1984; p. 169-176. 
10. Galvin S., Loomis C., Manabe M., Dhonailly D., Sun T.T.: 
The major pathways of keratinocyte differentiation as defined by 
keratin expression: an overview. Adv. Dermatol. 1989; 4: 
277-300. 
11. Coulombe P.A., Hutton M.E., Letai A. et al .: Point mutations 
in human Keratin 14 genes of Epidermolysis bullosa simplex 
patients. Cell 1991; 66: 1301-11. 
12. Bonifas J.M„ Rothman A.L., Epstein E.H. Jr.: Epidermolysis 
bullosa simplex: evidence in two families for keratin gene 
abnormalities. Science 1991; 254: 1202-1205. 
13. Lane E.B., Rugg E.L., Navsaria H. et al. : A mutation in the 
conserved helix termination peptide of keratin 5 in hereditary 
skin blistering. Nature 1992; 356: 244-246. 
14. Vassar R., Coulombe P.A., Degenstein R. et al.: Mutant 
keratin expression in transgenic mice causes marked abnormalities 
resembling a human genetic skin disease. Cell 1991; 64: 365-85. 
15. Yamamoto A., McGrath J.A., Judge M.R., Leigh I.M., Lane 
E.B., Eady R.A.J.: Selective involvementofkeratin 1 andkeratin 
10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis 
(bulous congenital ichtyosiform erythroderma). J. Invest. Derm. 
1992; 99/1: 19-26. 
16. Compton Digiovan J.J., Santucci S.K., Kearus K.S ., Amos 
C.L., Abangan D.L., Korge B.P., McBride O.W., Steinert P.M., 
Bale S.J .: Linkage of epidermolytic hyperkeratosis to the Type II 
gene cluster on chromosome 12q. Nat. Genet. 1992; 1/4: 
301-305. 
17. Rothnagel J.A., Dominey A.M., Dempsey L.D., Longley 
M.A., Greenhal D.a., Gagne T.a., Hubner M. , Frenk E., Hohl D., 
Roop D.R.: Mutations in the rod domains ofkeratin-1 andkeratin 
10 in epidermolytic hyperkeratosis. Science 1992: 257 (5073): 
1128-1130. 
18. Cheng J., Syder A.J., Yu Q. C., Letai A., Paller A.S., Fuchs 
E.: The genetic basis of epidermolytic hyperkeratosis - a disorder 
of differentation specyfic epidermal keratin genes. Cell 1992; 
70/5: 811 -819. 
19. Mieschke D., Wille G., Wild G.A.: Allele frequencies and 
segregation of human polymorphic keratins K4 and K5 . Am. J. 
Hum. Genet. 1990; 46: 548-552. 
20. Mieschke D., Wild A.G.: Polymorphic keratins in human 
epidermis. J. Invest. Dermatol. 1987; 88: 191-197. 
21. Lersch R., Stellmach V., Stocks C., Ginidice G., Fuchs E.: 
Isolation, sequence and expression of a human keratin K5 gene: 
transcriptional regulation of keratins and insights into pairwise 
control. Mol.Cell.Biol. 1989; 9: 3685-3697. 
22. Lessin S.R., Huebner K., !sobe M ., Croce C.M., Steinert 
P.M.: Chromosomal mapping ofhuman keratin genes: evidence 
of nonlinkage. J. Invest. Dermatol. 1988; 91: 572-578. 
23. Miller S.A., Dyckes D.D., Polesky H.P.: A simple s!'Jting out 
procedure for extracting DNA from human nucleatedcells. Nucl. 
Acids Res. 1988; 16: 1215. 
24. Southern E.M.: Detection of specific sequences among 
fragments separated by gel electrophoresis. J. Mol.Biol. 1975; 
98:503-517. 
25 . Sambrook J., Fritsch E.F„ Maniatis T.: Molecular Cloning -
A Laboratory Manual . Cold Spring Harbor Laboratory Press, 
New York 1989; p. 10.13-10.14. 
26. Wild G.A., Mieschke D.: Variation and frequency of 
cytokeratin polypeptide patterns in human squamous non-
keratinizing epithelium. Exp. Cell Res. 1986; 162: 114-126. 
27. Korge B.P., Song-Quing G., McBride W. Mieschke D., 
SteinertP.M.: Extensivesize polymorphism ofthe human Keratin 
lOchain residuesin the C-terminal V2 subdomain due to variable 
numbers and sizes of glycine loops. Proc.Natl.Acad.Sci.U.S.A. 
1992; 89:910-914. 
Authors' Addresses 
Siniša Tomič, chemist, Lastovska 4 , 41000 Zagreb, Croatia. 
Radovan Komel, Ph.D., professor of biochemistry, Head of the Medica! Center for Molecular Biology, 
Institute of Biochemistry, Medica! Faculty, Vrazov trg 2, 61000 Ljubljana, Slovenia. 
Aleksej Kansky M.D., Ph.D., professor of dermatology, Štihova 26, 61000 Ljubljana, Slovenia. 
Miroslav Blumenberg, Ph.D., professor of biochemistry and dermatology, R.O. Perlman, Departrnent ofDermatology, NYU 
Medica! Center, 550 First Ave 10016, New York, N.Y., U .S.A. 
118 acta dermatovenerologica A.P.A. Vol I, 92 , No 4