ARTICLE
Auteur(s) : Wenhua
Feng, Weitian Han, Xiaohui Man, Miao Jiang, Chaoying Bian, Ge
Wang, Xuefu Li, Dongxu Yi, Jianxin Li
Key Laboratory of Reproductive Health of Liaoning, 10 PuHe
Street, Huanggu District, Shenyang, 110031, China
accepté le 24 Février 2008
Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal
dominantly inherited disease which was first described by Vörner in
1901 [1]. It is characterized by diffuse yellow symmetric
hyperkeratosis, sharply bordered by erythematous margins, over the
entire surface of the palms and soles that appears 3 to 12 months
after birth. Reis and colleagues initially mapped EPPK to
17q12-q21, the locus of the type I acidic keratin cluster [2]. The
KRT9 gene, one of the type I keratin genes, is the only keratin
gene that is expressed solely in the terminally differentiated
epidermis of palms and soles [3, 4]. More recently, more than 20
different mutations in exon1 of the KRT9 gene have been identified
as responsible for EPPK [5-9]. The mutation in codon 161 of the
KRT9 gene was first reported in a French family by Torchard and
colleagues in 1994 [6]. To date, 5 different mutations in this
codon have been described as responsible for EPPK [5-7, 10, 11]. We
present the first family in Northeast China affected by EPPK and
carrying the heterozygous N161S mutation in the KRT9 gene. This
finding, together with the previous reports of the position 161
mutation of the KRT9 gene in different kindreds around the world
strongly suggests that position 161 of the KRT9 gene also
represents a mutation “hotspot” for EPPK.
Materials and methods
Family investigation
We examined and ascertained a five-generation family from Chaoyang
district of Liaoning province in Northeast China, including 16
affected and 33 unaffected individuals. The clinical diagnosis of
the autosomal dominantly inherited disease EPPK was performed in
the Affiliated Hospital of China Medical University, based on
dermatological and auxiliary examination. Peripheral blood samples
were collected from the participants, who gave their informed
consent, and were used for DNA extraction.
Genomic DNA amplification
Genomic DNA was extracted from peripheral blood by standard
techniques. A 436 bp fragment containing most of exon 1 of the KRT9
gene was amplified by polymerase chain reaction (PCR) using the
sense primer: 5′-TTGGCTACAGCTACGGCGGAGGAT-3′ and anti-sense primer:
5′-TGGTCCTTGAGATCATCAATAGTG-3′. These primers for previously
reported mutation hot spots were designed according to the KRT9
sequence [GI: 55956898]. The PCR program used consisted of
incubation at 94 °C for 5 min; followed by 35 cycles of
94 °C for 30 s, 60 °C for 1 min, 72 °C for
1 min; and then 72 °C for 5 min; reserved at
4 °C. PCR products were identified by 2% agarose gel
electrophoresis.
DNA sequencing
PCR products were purified using a DNA fragment purification kit
(TaKaRa Biotechnology Co., Ltd.) according to the manufacturer’s
recommended protocol. Sequencing processes were performed in
Shanghai at GeneCore BioTechnologies Co., Ltd. The sequencing
results were contrasted with sequences from the Genebank using the
biological software Chromas.
Restriction endonuclease analysis
In order to identify the 161 codon AAT → AGT mutations (N161S) of
KRT9, we did further analysis using the biological software Primer
Premier5 for restriction of the endonuclease screening function. It
was found that the codon 161 (N161S) mutation causes a new
endonuclease DdeI restriction site in the vicinity of the 548A>G
transition spot, the site is as follows: 5 ’- C ↓ TNAG-3′, 3 ’-GANT
↑ C-5′. And it does not exist in the wild-type KRT9 gene. With the
endonuclease digestion of the PCR product (436bp), the wild-type
KRT9 gene (without mutation) can not be cut into two fragments,
however with the 548A>G mutations, the restriction endonuclease
DdeI digestion of the PCR product could generate two fragments of
273 bp and 163 bp. DdeI endonuclease (Promega) was used to digest
the PCR products according to the manufacturer’s recommendations.
Then we detected the results using 8% polyacrylamide gel
electrophoresis.
Results
Family investigation
The pedigree comprised 49 individuals with 16 affected individuals,
exhibiting an autosomal dominant disease inheritance (figure 1). All the
affected members in the family have similar typical symptoms of
epidermolytic palmoplantar keratoderma (figure 2). The onset time
of the disease is different in different patients. Most of the
patients had symptoms of EPPK within 1 year after birth. The
proband (IV-10) showed a diffuse yellow palmoplantar keratoderma
with erythematous margins. Affected persons in all five generations
had severe epidermolytic hyperkeratosis of a yellowish appearance,
surrounded by a characteristic erythematous border, which was
completely confined to the palmoplantar surfaces. The nails were
not involved. Further clinical examination did not show any other
abnormalities.
DNA sequencing
Most coding sequences of exon 1 of the KRT9 gene, including the
hotspots reported previously, were amplified by PCR. Subsequent
sequencing of a patient’s (V5) DNA fragment revealed one
mutation in exon 1 of the KRT9 gene. A base substitution from A to
G at nucleotide 548 (548A>G) leading to a substitution of
asparagine by serine at codon161 (N161S) was found. This mutation
was confirmed by bi-directional sequencing (figure 3). The results
showed that patient is the heterozygote of a mutated gene.
Restriction endonuclease analysis
The results of polyacrylamide gel electrophoresis showed that all
five affected family members clearly show three bands of two
digestion fragments (237bp and 163bp) in addition to the wild type
undigested fragment (436bp) after the PCR products digested by
restriction endonuclease DdeI. However, all the healthy people in
the family lack these two digestion fragments. The digestion
fragments also cannot be detected in unrelated healthy individuals
(figure 4). The
results indicated that all affected family members have the N161S
mutation and they are all heterozygotes of mutated gene KRT9 while
the healthy individuals only have wild-type AAT.
Discussion
Palmoplantar keratodermas (PPKs) form a group of heterogeneous
diseases characterized by marked thickening of the epidermis on the
palms and soles. There are three clinical patterns: diffuse, focal
with extensive hyperkeratosis at point of friction, and punctuate
[12]. Of diffuse PPKs, epidermolytic PPK is probably one of the
most common keratin diseases with the prevalence of 4.4-5.2 per
100,000 among diverse populations from different regions around the
world [13, 14]. The group of PPKs with a diffuse pattern is also
rather heterogeneous. Within the first year of life diffuse
keratoderma may start with a focal pattern, but later on a
confluent hyperkeratosis is present. As the symptoms and clinical
descriptive classifications of PPK are complicated, mapping and
identification of genes are important for PPKs. In recent years the
more descriptive classification of keratodermas has switched to an
exact molecular genetic view, where gene functions are considered.
So after establishing a clinical diagnosis of EPPK, confirmation is
sometimes needed by means of documentation of biochemical defects
or gene mutation. In the future, molecular genetic classification
of keratodermas by gene functions will more and more replace the
traditional classification schemes [15].
This is the first EPPK kindred reported in Northeast China. In
China several EPPK kindreds and gene defects have been reported
recently and three novel mutations: L160F, N160H and Y166delinsWL
[8, 11, 16] were found. Kindreds that were previously reported
originate in the south and the middle of China [8, 11, 16, 17].
China is a very vast country with numerous nationalities, there are
obvious genetic differences between the northern and southern
populations. The EPPK kindred we present is the first to be
reported in Northeast China, which brings valuable information for
EPPK research in the Chinese population.
According to new versions of the sequence of keratin 9 published
after April 17, 2001 [GI: 13653405], a threonine at position 12 in
old versions was changed to serine and arginine. Our sequencing
supports this correction. In this article, we describe an amino
acid position in keratin 9 according to the sequence published on
June 26, 2007 [GI: 55956898], so position 161 of keratin 9 in our
article is the same as position 160 in previously published
research articles.
The Vörner form of palmoplantar keratoderma may be distinguished
from other forms by the early onset and diffuse pattern of thick
yellowish hyperkeratosis in the absence of associated features
[18]. The disorder usually affects the hands, which are relatively
spared in non-EPPK, and histologic examination reveals
epidermolytic changes [19]. Here, we diagnosed one Northeast
Chinese family on the basis of these criteria, as part of a PPKs
study in this country and we have identified the disease-causing
mutation, N161S, in all affected family members, occurring within
the coil lA segment of the rod domain of KRT9. As EPPK is part of a
group of heterogeneous symptoms, one certain mutation may be
associated with divers additional symptoms such as breast and
ovarian cancer and knuckle pads [6, 8, 17, 20]. All the affected
members in this family have similar typical symptoms of
epidermolytic palmoplantar keratoderma without knuckle-pad-like
lesions. The nails are not involved. Further clinical examination
did not show any additional abnormalities or associated diseases.
However, the EPPK family with the same N161S mutation of KRT9 which
Zhang BR and colleagues [17] reported in Central Southern China
(Zhejiang Province) showed us additional phenotypes (knuckle pads
and nail lesions). It is worth mentioning that an EPPK family
caused by the L160F mutation with similar additional phenotypes,
which Lu Y and colleagues [8] reported, also occurred in Central
Southern China (Shandong Province). These are the only two EPPK
cases with the knuckle-pad-like lesions reported in this area so
far. Is it a coincidence? Or do geographical origins play some role
in the characteristics of the phenotype? Further studies on these
cases may cast new light on the function of Keratin 9.These results
are important contributions to the investigation of the
genotype/phenotype correlation and afford molecular genetic
knowledge for future clinical diagnosis and gene therapy of
EPPK.
Keratins are a group of proteins that form the intermediate
filament cytoskeleton of epithelial cells, which are important for
structural integrity. In keratoderma, excessive production of
normal or altered keratin on the palms and soles is found. Keratin
9 is only found in palmoplantar skin and keratin analysis in
epidermolytic hereditary palmoplantar keratoderma showed mutations
in keratin 9 [7, 12]. Numerous additional investigations have
confirmed mutations in keratin 9 in patients with EPPK [21]. Codon
161 is frequently altered in EPPK reports to date, in different
kindreds from diverse regions around the world [5, 6, 10, 22-24].
Here we report a family in Northeast China with EPPK carrying the
heterozygous N161S mutation in the KRT9 gene. Our findings together
with the worldwide presence of the mutation in codon 161 of KRT9,
strongly suggest that the position 161 represents a “hot-spot” for
mutation in KRT9 gene for EPPK.
Acknowledgements
We would like to thank all of the volunteers who participated in
this study, especially the individuals with EPPK and their
families. Thanks also to the specialists in Affiliated Hospital of
China Medical University who performed the diagnosis for the EPPK
patients.
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