ARTICLE
Auteur(s) :, Alessandro TERRINONI2, Barbara
COCUROCCIA1, Emanuela GUBINELLI1, Giovanna
ZAMBRUNO1, Eleonora CANDI3, Gerry
MELINO2,3, Giampiero
GIROLOMONI1,*
1II Dermatological Division, Istituto Dermopatico
dell’Immacolata, IDI-IRCCS, Rome, Italy
2Laboratory of Biochemistry IDI-IRCCS c/o Department of
Experimental Medicine and Biochemical Sciences, University of Rome
Tor Vergata, Rome, Italy
3Department of Experimental Medicine and Biochemical
Sciences, University of Rome Tor Vergata, Rome Italy
accepté le 3 Août 2004
Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal
dominant skin disorder characterized by well-demarcated, symmetric
hyperkeratosis of the palms and soles associated with histologic
findings of hyperkeratosis and epidermolysis (ballooning
degeneration) starting in the spinous layer [1-3].
Ultrastructurally, there is vacuolization of the cytoplasm and
abnormal keratin filament network characterized by tonofilament
clumping [4]. EPPK was initially mapped to 17q12-q21, the locus of
the type I acidic keratin cluster [5]. Subsequently, mutations were
detected in the keratin 9 (KRT9) gene, which is expressed
exclusively in the differentiating skin of the palms and soles.
These mutations generally affect the highly conserved coil 1A
region of the alpha-helical rod domain of KRT9, a domain thought to
be important for keratin heterodimerization [6]. To date a number
of KRT9 gene mutations responsible for EPPK have been described.
The most common is the substitution of arginine in position
162 with tryptophan (R162W), which has been reported in North
American, European and Japanese patients [4, 6-11].We present the
first three Italian families affected by EPPK carrying the
heterozygous R162W mutation in the K9 gene. This finding confirms
that position 162 represents a mutation “hot-spot”, probably
in relation to the peculiarity of the sequence.
Patients
In this study we investigated three unrelated Italian families from
different geographical areas of Italy (Family A: North-West; family
B: South; family C: Center), whose members were diagnosed as having
EPPK based on the characteristic clinical and histopathological
features. All the probands from the families studied showed diffuse
thickening of palms and soles with a yellowish discoloration,
surrounded by a well demarcated erythematous border. No other body
sites were affected. Oral mucosa, hair and nails were normal. The
proband from family A (40-year old, male; ( Figure 1 )) referred
the onset of palmoplantar erythema a few months after birth. His
1-year-old daughter presented hyperkeratosis of palms and soles. No
other family member was affected. The proband from family B
(35-year old, female) reported the presence of a diffuse
palmoplantar erythema at birth, with absence of neonatal fragility
or blistering. Hyperkeratosis appeared after the first month of
age. She had persistent PPK throughout her life with cycles of
improvement and worsening. Patient siblings displayed a similar
phenotype, with involvement limited to the palms and soles; similar
clinical features were reported for all the affected members of the
family (( Figure 2 )). The
proband from family C (40-year old, male) was told that his
palmoplantar hyperkeratosis appeared after the first month of age
and subsequently ran a chronic relapsing course. His sister and
father presented the same skin changes.
Methods and results
Biopsy samples from all the three probands showed marked
hyperkeratosis, vacuolar degeneration of keratinocytes in the upper
spinous and granular layers, pyknotic nuclei, and a thickened
granular layer containing an increased number of keratohyaline
granules (( Figure 3 )).
Transmission electron microscopy of the affected palmar skin of
probands from family A and C showed epidermolysis, cytoplasmic
vacuolization and marked tonofilament clumping in suprabasal cells.
To screen the patients for the presence of mutations in the KRT9
gene, we analyzed the mRNAs derived from hyperkeratotic skin by
reverse transcription polymerase chain reaction (RT-PCR) analysis,
followed by direct sequencing. Total RNA was extracted from two
3-mm skin biopsies of the palmar region, using the Qiagen RNeasy
mini kit (Qiagen, Milano, Italy). RT-PCR reactions were performed
by using the RT-PCR One Step System (Invitrogen, San Giuliano
Milanese, Italy) with 100 ng of total RNA, according to the
manufacturer’s instructions. The entire coding region of the KRT9
gene was amplified with the primers K9F1 (5′-AGC CGG TAG CAC TCC
TAT CAC TGC TT-3′, + strand) and K9R5 (5′-GAC CAC TGG TTC TAC TCT
GTT TTC C-3′, - strand). The following PCR program was used: 51°C
for 60 minutes (cDNA synthesis) and 94 °C for
2 minutes; followed by 40 cycles of: 94 °C for
35 seconds, 59 °C for 2 minutes and 30 seconds,
and 70 °C for 30 seconds. PCR fragments were gel purified
with the Qiaex II extraction kit (Qiagen) prior to sequencing. In
all cases, PCR products were directly sequenced using the
amplification primers and additional internal primers.
Approximately 100 ng of purified template DNA was sequenced
with the BigDye Termination Reaction kit (Perkin Elmer, Roma,
Italy) on an ABI-PRISM 377 DNA sequencer (Perkin Elmer)
according to the manufacturer’s protocols. The sequence of the
coding region of KRT9 gene revealed the heterozygous 484C→T [12]
transversion (( Figure 4 )), leading
to the substitution of a highly conserved arginine in position
162 with a tryptophan. The substitution is a non-conservative
change of a basic (arginine) with a neutral amino acid
(tryptophan). To confirm the mutation, we sequenced the exon
1 of keratin KRT9 gene from genomic DNA, extracted from
patients’ blood according to standard protocols. The probands and
all affected members of the respective families have been analyzed
by sequencing the mutation region with forward and reverse primers.
This analysis confirmed the presence of the mutation in all
affected individuals.
Discussion
PPKs are a group of clinically and genetically heterogeneous
diseases characterized by the presence of marked hyperkeratosis and
thickening of the epidermis of the palms and soles [13]. The
classification was originally based only on clinical criteria such
as morphology of the skin lesions, inheritance pattern and the
selective involvement of different body sites [14]. Recently, the
classification has been modified to include histopathological
findings and when known, molecular defects. PPKs are now subdivided
into four major classes [15, 16]: diffuse PPKs, focal PPKs,
punctate PPKs and palmoplantar ectodermal dysplasias. Diffuse PPKs
can be further subdivided into epidermolytic PPK (Voerner type,
EPPK; OMIM #144200) and non-epidermolytic PPK (Unna-Thost, NEPPK;
OMIM #148400). Both types were described clinically in the
beginning of last century, but the molecular defects have only been
identified in the last decade [16]. These forms are inherited as
autosomal dominant disorders with variable penetrance. EPPK is
clinically characterized by a diffuse thickening of the skin of the
palms and soles with an erythematous border. Peeling and blistering
can also be present. Histologically, the affected skin shows
hyperkeratosis and vacuolar cytolysis of the granular layer.
Electron microscopy shows vacuolization of keratinocytes of the
granular layer and clumping of keratin filaments. NEPPK has similar
clinical presentation to EPPK, but the lesions usually lack an
erythematous border. Histologically, NEPPK lesions show
orthokeratosis and acanthosis, but no vacuolar degeneration.
Mutations in different keratin genes have been shown to underlie
distinct types of PPKs. Interestingly, different mutations of the
same keratin have been associated with completely distinct
phenotypes [16-18]. Keratins and their attachment complexes
represent the major structural cytoskeleton in keratinocytes and
are essential for maintaining the integrity of the epidermis.
Keratins are divided into two groups, type I acidic keratins
(K9–K20) and type II basic keratins (K1–K8); genes of the first
group are localized on chromosome 17 (17q12–q21), genes codifying
for the second one are on chromosome 12 (12q11–q13). Keratins
contain a central coiled-coil rod domain with four alpha-helical
segments (1A, 1B, 2A, and 2B) separated by three non-helical linker
elements (L1, L12, and L2) [19, 20]. Non-helical head and tail
domains flank the rod domain. The rod domain starts and ends with
two short, highly conserved, amino acid sequences known as the
“helix initiation peptide” (HIP) and the “helix termination
peptide” (HTP). The HIP and HTP represent the “helix boundary
motifs” [21, 22]. Keratin HIP and HTP interaction leads to
dimerization to form a basic-acidic heterodimer [22]. Physical
interactions between primary heterodimers give rise to the
10 nm fibril, the major component of intermediate filaments
(IF). Further interactions contribute to form structures of higher
order, leading to the formation of the IF network, typical of
epidermal keratinocytes and all epithelial tissues. The IF network
is responsible for the typical impermeability and capacity to
resist to mechanical stresses of epidermis [20]. Mutations located
within the rod domain, and mainly within HIP and HTP, cause
keratin-dependent skin diseases because they interrupt or interfere
with the formation of the initial dimer, and perturb the stability
of IF network [23]. 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 [24]. Keratin pairs are
expressed in a tissue and differentiation specific pattern, with K5
and K14 expressed in the basal layer of the epidermis, and K1 and
K10 in suprabasal cells. The cells of upper spinous and granular
layers of the epidermis also express small amounts of keratin K2e
[25]. The type I keratin, K9, is expressed in the suprabasal layer
selectively in palmar and plantar skin [26]. Mutations in different
keratin pairs manifest with distinct phenotypes, for example
mutations in K5 or K14 cause epidermolysis bullosa simplex, and
mutation in K1 or K10 cause epidermolytic hyperkeratosis [27].
Mutations in KRT9 have been associated with EPPK, Voerner type
[5, 28, 29]. To date more than 30 mutations in keratin K9 have
been found in patients affected by EPPK [Human IF Database
http://134.36.196.124/interfilwlcm.htm]. The missense mutation
R162W is the most frequent alteration reported in EPPK to date in
different kindreds [4, 6-11]. The high frequency of mutation
involving the first timine of codon 162 could to be due to
ectopic crossing-over during meiosis of gametogenesis. This
phenomenon can be related to the peculiar structure/sequence of the
HIP of the KRT9 gene. Further evidence to support this hypothesis
is the observation that many of the R162W mutations described to
date arise in sporadic patients, demonstrating that the worldwide
presence of the mutation is not due to the spreading of the mutated
allele by population migration, but to de novo mutations. Here we
report on three Italian unrelated families with EPPK carrying the
heterozygous R162W mutation in the KRT9 gene. Our findings together
with the previous reports, strongly suggest that the position
162 represents a “hot-spot” for mutation in KRT9 gene,
probably due to the peculiarity of the sequence.
Acknowledgements
Financial support: The work was supported by the Italian Ministry
of Health and by a Telethon grant GGPO2251 to Eleonora Candi.
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