ARTICLE
Auteur(s) : Andrea Codispoti11, Enrico Colombo12, Loredana Zocchi1,
Valeria Serra1, Ginevra Pertusi2, Giorgio
Leigheb2, Rossana Tiberio2, Guido
Bornacina2, Riccardo Zuccoli2, Antonio
Ramponi3, Elena Campione4, Gerry
Melino1, Alessandro
Terrinoni1
1IDI-IRCCS Biochemistry laboratory c/o Dep.
of Experimental Medicine, University of Tor Vergata, Via
Montpellier, 1. 00133 Rome, Italy
2Dermatological Clinic, University of Piemonte
Orientale ‘A. Avogadro’, Novara, Italy
3Service of Pathological Anatomy, University
of Piemonte Orientale ‘A. Avogadro’, Novara, Italy
4Department of Internal Medicine, Dermatology
Clinics, University of Rome ‘Tor Vergata’
accepté le 20 Octobre 2008
The autosomal dominant epidermolytic palmoplantar keratoderma
(EPPK, OMIM#144200) Vörner-type is a skin disorder characterized by
diffuse, yellow thickening of the skin on the palms and soles [1]
associated with histological findings of hyperkeratosis and
epidermolysis starting in the spinous layer [2-4]. This
characteristic histological feature can be found in other skin
diseases such as epidermolytic hyperkeratosis or bullous congenital
ichthyosiform erythroderma, linear epidermal nevus, solitary or
disseminated epidermolytic acanthoma and as an incidental and
non-specific pattern in different cutaneous lesions [5-7].
Mutations responsible for EPPK have been demonstrated in the
keratin K9 gene, KRT9 [4, 8, 9], which is expressed exclusively in
the differentiating skin of palms and soles [10]. Keratins are
members of the keratin intermediate filaments (KIF) superfamily and
provide structural integrity to epithelial cells [11]. Structurally
all keratins consist of a central α-helical rod domain of 310 amino
acids flanked by non-helical head (V1) and tail (V2) domains [12,
13]. The rod domain is divided into the 1A, 1B, 2A and 2B domains
and each one consists of heptads repeats of hydrophobic amino
acids, which provide a hydrophobic seal on the helical surface,
enabling the coiled-coil structure between two keratin polypeptides
[14]. The start and end of the rod domain are two short regions:
the helix initiation motif of the 1A helical segment at the amino
terminus and the helix termination motif of the 2B helical segment
at the carboxyl terminus [13]. The helix initiation motif comprises
the first 15 residues of the 1A segment and is highly conserved in
all intermediate filament types. The sequences of these regions
play very important roles in filament assembly [12].
To date, various KRT9 mutations, have been described in EPPK [8,
15-20], generally affecting the highly conserved coiled 1A region,
of the alpha-helical rod domain [18]. The most common mutation
found in EPPK is the substitution of an arginine in position 163
with a tryptophan (R163W), which has been reported in North
America, European, Japanese and Italian populations [1, 4, 18, 19].
In some rare cases, EPPK is associated with knuckle pad formation,
a benign thickening of the knuckle epidermis [17, 21-23]. This
additional phenotype however cannot be associated, at present, to a
specific group of K9 mutations. In addition, to date, only a few
cases of EPPK with involvement of the dorsal region of the finger
joints, called knuckle pad-like keratosis, have been published in
the literature, but these, however, are associated with different
mutations of the KRT9 gene [16, 17, 24-26]. In this paper, we show
that the knuckle epidermis of EPPK patients carrying the R163W
mutation shows over-expression of wt and mutant K9, whereas knuckle
epidermis of normal controls does not. These results provide a
plausible mechanism for a knuckle pad phenotype.
Materials and methods
Light microscopy
Ethical approval and informed consent, according to the Helsinki
declaration, was obtained before the skin biopsies were collected.
Biopsy samples from patients were processed for light microscopy
and samples were paraffin embedded and stained using
hematoxylin-eosin according to standard methods.
Molecular genetic analysis
Total RNA was extracted from two 3-mm skin biopsies of the palmar
region, using the Qiagen RNeasy mini kit (Qiagen, Milano, Italy).
cDNA synthesis using the ImProm-II Reverse Transcription System Kit
(Promega, Madison, WI, USA). For PCR analysis, the KRT9 gene from
the 5’-UTR to 3’-UTR was amplified using oligonucleotide pairs:
K9-F1, 5’-AGC CGG TAG CAC TCC TAT CAC TGC TT-3’; K9-R5, 5’-GAC CAC
TGG TTC TAC TCT GTT TTC C -3’. PCR was performed using Platinum Taq
DNA Polymerase High Fidelity (Invitrogen, Invitrogen, Carlsbad, CA,
USA). PCR products were directly sequenced using the amplification
primers and additional internal primers. Approximately 100 ng
of purified template DNA was used for sequence analysis. The allele
specific digestion analysis was performed amplifying a region of
128 bp, containing the mutated sequence, from cDNA samples. PCR was
performed using primers pairs, K9-F Sty,
5’-ACCATGCAGGAACTCAATCCT-3’, K9-R2,
5’-TGGATAGGAGCAGGTCCCTTCTTGTC-3’. The forward primers containing a
mismatch base (underlined) that together with the 487C->T
tranversion create a new Sty-I restriction site
CT↓CTTCG↑G only in the cDNA
amplified from the mutated allele. The Sty-I restriction of this
region generates two fragments of 107 and 21 bp, with no effect on
the wt 128 bp amplycon.
RTqPCR
Real-time PCR was performed on an ABI-7500 SDS instrument (Applied
Biosystem, Foster City, CA, USA) usind the Platinum SYBR Green qPCR
SuperMix UDG with ROX (Invitrogen Carlsbad). The PCR reactions were
performed using primers TGG CTA TGG GAG TGG GTT TGG (+), and GCA
GCA GGT CCC TTC TTT GCT (-), according standard ABI protocol. Actin
has been used for internal standard control, with primers AAA GAC
CTG TAC GCC AAC A (+), CGG AGT ACT TGC GCT CAG (-).
Results
We investigated a southern Italian family affected by PPK, 11 of
the 24 members of the family showed clinical symptoms and the
pedigree was consistent with an autosomal dominant transmission
(figure 1A).
The clinical analyses of three of the affected members of the
family, show a diffuse palmoplantar keratoderma with a
well-demarcated erythematous border. In all patients the lesions
initially appeared around the 2nd month of age (figure 1B, C). Only
patient III-6 showed hyperkeratotic knuckle pad-like plaques on
proximal interphalangeal joints and on metacarpophalangeal joints,
more severe on the more frequently used right hand (figure 1D, E). None of the
patients studied presented any involvement of hair, teeth and
nails.
Histopathological analysis of a biopsy from palmar skin of
patient III-6 showed the presence of hyperkeratosis with
epidermolysis of the suprabasal layer (figure 1F), characteristic
of EPPK. To further investigate the nature of the knuckle pad
lesions, we collected a skin biopsy from patient III-6.
Histological analysis (HE) of this sample showed hyperkeratosis and
epidermolysis, with vacuolar degeneration of keratinocytes in the
upper spinous and granular layers (figure 2A, asterisks; B,
white arrows), pyknotic nuclei (figure 2B, gray arrows),
and a thickened granular layer containing an increased number of
keratohyaline granules (figure 2B, black arrows).
This evidence, together with the clinical features, was compatible
with a diagnosis of Vöerner type EPPK.
Patients III-6 and IV-6 (figure 1A) were analysed
for the presence of mutations in the keratin 9 gene. RNA was
extracted and retro-transcribed from a biopsy of palmar
hyperkeratotic skin, and the coding sequence of the KRT9 gene was
entirely sequenced. The sequence analysis showed a mutation located
in exon 1, involving the helix initiation motif (1A domain). This
mutation has already been described in literature [4, 9], and is
characterized by a heterozygous 487C→T transversion (figures 3A, B) leading to
the non-conservative substitution of a highly conserved arginine
(basic) in position 163 with a tryptophan (neutral). To confirm the
presence of the mutation, we sequenced exon 1 of keratin K9 gene
from genomic DNA, extracted from peripheral blood lymphocytes. The
analysis confirmed the presence of the mutation in all affected
individuals (II-6, III-6, IV-6; data not shown).
In addition, we collected a biopsy from the knuckle pads of
patient III-6 and from the same region of an unaffected unrelated
donor, to evaluate keratin 9 expression. qRT-PCR analysis showed a
significant K9 expression in the knuckle pad of patient III-6
compared to the control (figure 3C). This indicates
an abnormal expression of K9 in the dorsal region of the patient’s
fingers. To demonstrate if the K9 expressed by the patient was wt
or mutant, we established a specific assay. A cDNA fragment
containing the 128 bp region of the mutation was amplified by PCR,
using a forward primer containing one mismatch base, that together
to the C>T mutation creates a new Sty-I restriction site in the
mutant allele (see methods for details). The digestion of the
fragment with Sty-I, gave rise to two visible bands from the
patient’s cDNA sample (figure 3D, lane 1), due to
the presence of both the wt (128 bp) and the mutated allele (107
bp, black arrow), the second small 21 bp fragment is not visible.
Only the undigested, full length, band is present in the wt control
(figure 3D, lane
2), thus demonstrating the expression of the mutated allele in the
dorsal region of finger.
Discussion
Keratins are members of the keratin intermediate filaments
(KIF) superfamily and are essential for the structural integrity of
epithelial cells. The helix initiation motif is highly conserved in
all intermediate filament types. Dominant-negative mutations in
keratin genes, largely affecting the central α-helical domain,
result in disorders characterized by epithelial fragility and/or
hypertrophy. The distribution of the lesions in skin diseases
reflects the expression pattern of the affected keratin [27].
Keratin pairs are expressed in a tissue and differentiation
specific pattern [11]. Almost 50% of the keratin mutations affect
the arginine residue at position 10 of 1A domain of type I
keratins. In the case of KRT9, the 1A hotspot mutations are R163W
and R163Q, which appear to be the most common genetic defects
reported in EPPK to date [4, 8, 9, 15, 18, 20, 28]. This is a non
conservative change consistent with the methyl CpG deamination
mechanism of mutation, which accounts for approximately 90% of
human point mutations [29].
The KRT9 gene is strongly expressed in the palmoplantar granular
layer, above the primary epidermal ridges in the centre of the
papillary ridge [5, 11]. Raised papillary ridges overlying the
primary epidermal ridge receive most of the compression stress on
the skin [1]. This is also the region where the
palmoplantar-specific K9 gene is most highly expressed, strongly
suggesting that the function of K9 is to offer an extra
reinforcement in this stress-bearing epidermis. The palmoplantar
skin becomes abnormally thickened and hyperkeratotic in response to
physical stress, when K9 mutations weaken the cell cytoskeleton
[30].
Knuckle pads are well-circumscribed plaques, located in the skin
over the dorsal metacarpophalangeal and interphalangeal joints [23,
31]. Knuckle pads are known to be present in few cases of EPPK,
however the incidence of this clinical feature is probably
underestimated. Up to now, knuckle pads have been associated with
repetitive trauma related to certain sportive or work activities
[17]. Nevertheless, the histological analysis of knuckle pads from
our patient showed the presence of epidermolysis in the suprabasal
layer, a classical finding of all skin diseases caused by keratin
mutations, and localised in the specific layer of K9 expression.
Indeed, here we report for the first time that this gene is
ectopically over-expressed in the knuckle pads. qRT-PCR, performed
on dorsal skin biopsies, taken from both affected and healthy
donors, proved that a basal level of keratin 9 expression is
present in the control sample, but in the proband’s knuckle pads
there is an increase of almost 90-fold. Furthermore the presence of
the mutant allele has been demonstrated in the affected individual.
It has been previously described that the onset of these
hyperkeratotic plaques was due to mechanical friction, but our
proband denied manual labour. EPPK is usually associated to
epidermolysis in the suprabasal layers limited to palmoplantar
skin, but our findings strongly support the idea that the knuckle
pad formation is associated, in some patients, with a specific
transgrediens over-expression of the mutated K9 (R163W), together
with the wild type, and thus generating hyperkeratosis and
epidermolysis in region adjacent to, but outside of the
palmoplantar areas.
Acknowledgments
Financial support: We would to thanks to contract grant sponsor:
EU-Grants EPISTEM (LSHB-CT-019067), FIRB-Grants RBNE01KJHT_004,
RBNE01NWCH_008; MIUR/PRIN 004064744_003; AIRC rif. 1338; ISS n.
530/F-A19. The work was also supported by Grant Telethon GGPO4110
to Gerry Melino; ISS ‘Programma Italia Usa’, N526D5; ISS
RF06-Conv.73.2. Conflict of interest: None.
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