1. health and fitness
2. disease

OBSERVATION
Diffuse Nonepidermolytic Palmoplantar
Keratoderma Caused by a Recurrent
Nonsense Mutation in DSG1
Hannah Keren, BSc; Reuven Bergman, MD; Mordechai Mizrachi, BSc;
Yechiezkel Kashi, PhD; Eli Sprecher, MD, PhD
Background: Mutations in genes coding for 2 desmo-
somal proteins, desmoglein 1 and desmoplakin, have been
shown to cause autosomal dominant keratoderma palmoplantaris striata.
Observations: We describe a family affected with a dif-
fuse nonstriated form of palmoplantar keratoderma. Histopathologic examination of skin biopsy specimens disclosed cell-cell disadhesion in the suprabasal layers of the
epidermis, as previously described in keratoderma palmoplantaris striata. We therefore genotyped all family
members using microsatellite markers encompassing 3
keratoderma palmoplantaris striata-associated loci. Hap-
I
Author Affiliations:
Department of Dermatology
and Laboratory of Molecular
Dermatology, Rambam Medical
Center, Haifa (Ms Keren,
Drs Bergman, Kashi, and
Sprecher, and Mr Mizrachi);
Faculty of Biotechnology and
Food Engineering (Ms Keren
and Dr Kashi), Bruce Rappaport
Faculty of Medicine
(Drs Bergman and Sprecher),
and Biotechnology
Interdisciplinary Unit
(Mr Mizrachi), Technion–Israel
Institute of Technology, Haifa.
Financial Disclosure: None.
lotype analysis suggested linkage of the disease to 18q12.1,
which harbors the DSG1 gene, encoding desmoglein 1.
Mutation analysis eventually led to the identification of
a causative recurrent nonsense mutation in this gene.
Conclusions: Mutations in DSG1 are not exclusively associated with striated palmoplantar keratoderma. The present study illustrates the efficacy of an integrative diagnostic approach to palmoplantar keratodermas involving
clinical assessment, pathologic examination, microsatellite marker screening, and mutational analysis.
Arch Dermatol. 2005;141:625-628
NHERITED PALMOPLANTAR KERAtodermas (PPKs) occur in a large
group of cornification disorders
characterized by extensive phenotypic heterogeneity.1 The Online Mendelian Inheritance in Man
(OMIM) catalogue (http://www.ncbi.nlm
.nih.gov/entrez/query.fcgi?db=OMIM)
mentions more than 35 genetic diseases
manifesting with prominent PPK. Over the
last several years, much progress has been
achieved toward a better understanding of
the molecular basis of these disorders. Mutations in more than 20 distinct genes have
been described in various forms of PPK.
Many of these genes code for structural
proteins (eg, keratins) or components of
the desmosomal plaque, which are all
known to play an important function during keratinocyte differentiation. 1 The
physiologic role of other molecules associated with the pathogenesis of PPK, such
as connexins and secreted LY6/UPAR–
related protein 1 (SLURP-1), is less well
understood.2,3
To overcome the difficulties posed by
phenotypic and genetic heterogeneity in
the diagnosis of inherited PPK, a number
of classification schemes have been de-
(REPRINTED) ARCH DERMATOL/ VOL 141, MAY 2005
625
vised in which morphologic features are
used to predict the underlying molecular
defect.4 For example, the association of
periodontitis and PPK is suggestive of Papillon-Lefevre syndrome caused by mutations in the cathepsin C gene,5 whereas the
coexistence of PPK and deafness is suggestive of a connexin gene mutation.2
Keratosis palmoplantaris striata (KPS)
is a rare autosomal dominant disorder
characterized by linear hyperkeratotic
streaks along the volar surface of the fingers and focal keratoderma over the soles.
Nonsense and frameshift mutations in
DSG1 and DSP encoding 2 desmosomal
proteins, desmoglein 1 and desmoplakin, demarcate 2 KPS subtypes, type I
(OMIM 148700) and type II (OMIM
125647), respectively.6,7 Recently, a frameshift mutation affecting the keratin 1 tail
domain was found to underlie KPS type
III (OMIM 607654) in a large kindred of
British extraction.8
Keratosis palmoplantaris striata is regarded as a prototypic desmosomal genodermatosis.9 Indeed desmoglein 1 and desmoplakin are critical components of the
desmosomal plaque in the upper epidermis, and frameshift mutations at the tail
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A
C
MUTATION ANALYSIS
B
Genomic DNA was PCR amplified with primer pairs covering
the entire coding sequence of the DSG1 gene as well as intronexon boundaries.13 Polymerase chain reaction amplification was
performed using Taq polymerase (Qiagen, Valencia, Calif) and
Q solution according to the manufacturer’s instructions. Gelpurified amplicons were subjected to bidirectional sequencing using Big Dye Terminator (PE Applied Biosystems). To verify
R26X, a 169–base pair PCR fragment, encompassing exon 2,
was PCR amplified and digested with BsiYI endonuclease.
D
RESULTS
CLINICAL FINDINGS
Figure 1. Clinical and pathological features. A, Diffuse thickening and
fissuring of the palmar skin and volar surface of the fingers. B, Plantar
keratoderma involving weight-bearing areas. C, A skin biopsy specimen from
the patient’s palmar skin demonstrates marked orthohyperkeratosis and
papillomatosis (hematoxylin-eosin, original magnification ⫻25). D, Higher
magnification demonstrates widening of the intercellular spaces and
disadhesion of keratinocytes in the spinous and granular cell layers
(hematoxylin-eosin, original magnification ⫻400).
domain of keratin 1 have been shown to affect the function of the desmosomal plaque during cornification.10
Thus, striated hyperkeratosis, as opposed to diffuse hyperkeratosis, is considered to be predictive of mutations
in genes coding for components of the desmosomal
plaque. In this article, we present the unusual case of a
family affected by a diffuse nonepidermolytic form of PPK
caused by a recurrent mutation in DSG1.
METHODS
PATIENTS
We obtained skin biopsy and blood samples after receiving informed and written consent from each participant according
to a protocol reviewed and approved by the local Helsinki Committee and by the Israeli Ministry of Health. Skin biopsy specimens were processed for regular histologic analysis as previously described.11 Blood was drawn from each individual (15
mL) and genomic DNA was isolated from blood samples using the salt chloroform extraction method.
MICROSATELLITE ANALYSIS
Ten polymorphic microsatellite markers (F13A1, D6S1564,
D6S309, D6S470, D12S368, D12S83, D12S1294, D18S877,
D18S1102, and D18S535) were selected from the GDB human
genome database (http://www.gdb.org), spanning each of the 3
gene loci previously shown to be associated with KPS.6-8 Genotypes of all individuals for each marker locus were established
by polymerase chain reaction (PCR) amplification of genomic
DNA using Supertherm Taq polymerase (Eisenberg Brothers Co,
Givat Schmuel, Israel). The PCR products were separated by polyacrylamide gel electrophoresis, run on an ABI 310 sequencer (PE
Applied Biosystems, Foster City, Calif) for fluorescently labeled
markers, or visualized using the silver staining method.12
(REPRINTED) ARCH DERMATOL/ VOL 141, MAY 2005
626
The proband was a 50-year old man of Jewish Yemenite
origin. From age 3 years, he had thickening of the skin
of his palms and soles accompanied by painful fissures.
Three of his children displayed a milder form of keratoderma, mainly evident on the soles. His grandparents and
parents were unavailable for examination or DNA sampling, but the proband indicated that his maternal grandfather, but not his mother, had reportedly been affected
by a similar disease. Treatment with etretinate for several months led to partial improvement of his condition
but was discontinued at the patient’s request because of
excessive skin dryness.
On examination, diffuse hyperkeratosis and fissuring of the volar surface of the hands and digits were observed (Figure 1A). Similar features were seen over
weight-bearing areas of the soles and toes (Figure 1B).
Mild onycholysis was accompanied by yellowish discoloration of most nails. Hair, teeth, mucosae, and nonpalmoplantar skin were normal.
Histologic examination of a skin biopsy specimen obtained from the palmar skin revealed some papillomatosis and marked orthohyperkeratosis in the epidermis
(Figure 1C). Widening of the intercellular spaces and disadhesion of keratinocytes were observed in the upper spinous and granular cell layers (Figure 1D).
HAPLOTYPE ANALYSIS
Since the histopathologic features observed in skin biopsy specimens from the patient resembled those previously described in KPS,6,7 we considered the possibility
that a mutation in a gene previously associated with this
disease might underlie the diffuse PPK displayed by the
proband. To assess this possibility, we established 3 panels of microsatellite markers spanning the 3-gene loci previously shown to be associated with KPS on 18q12.1
(DSG1), 6p24 (DSP), and 12q13 (KRT1).6-8 Markers were
selected based on their index of heterogeneity and short
distance to each of the 3 genes. We established the genotype of each of the 7 family members at the 3 loci. Haplotype analysis revealed that all affected individuals shared
a common 11.4-megabase chromosomal segment between markers D18S877 and D18S535 on 18q12.1, encompassing the DSG1 locus (Figure 2), which suggested the existence of a pathogenic mutation in this gene.
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R26X/WT
126 126
D18S877
D18S1102
94
126 130
92
90
148 146
D18S535
T
G
A
A
T
T
C
N
G
A
A
T
C
C
A
G
A
A
T
C
C
A
92
146 146
D18S877
126
126
126
126
126
126
126
126
D18S1102
92
90
92
90
94
90
94
90
D18S535
146
146
146
146
148
146
148
146
126 126
94
WT/WT
90
148 146
T
G
A
A
T
T
C
C
Figure 2. Haplotype analysis of 18q12.1, which harbors the DSG1 locus. All
affected individuals share a common haplotype (allele size in base pairs
indicated in the red boxes) over 11.4 megabases spanning the DSG1 gene
(arrows).
MUTATION ANALYSIS
We analyzed genomic DNA extracted from the patient’s
blood lymphocytes for pathogenic mutations in DSG1.
All exons and intronic boundary regions of the gene were
PCR amplified and directly sequenced. A single heterozygous C→T transition at complementary DNA position 76 (starting from the ATG) was identified in all affected individuals (Figure 3). This mutation results in
the substitution of a stop codon for an arginine residue
at position 26 of the amino acid sequence (R26X) and
has been previously described in a sporadic case of striated keratoderma.13 We developed a PCR–restriction fragment length polymorphism assay based on the fact that
the mutation abolishes a restriction site for BsiYI endonuclease (Figure 4). Using this assay, we confirmed segregation of the mutation in the family.
COMMENT
Desmosomal cadherins, which include a number of desmocollin and desmoglein isoforms, are transmembranal
proteins that play a critical role in cell-cell adhesion and
are part of pivotal signal transduction pathways regulating cell growth and differentiation.14 In accordance with
their pleiotropic functions, abnormal desmosomal cadherins have been linked to a growing number of inherited and acquired skin diseases.9 Desmoglein 1, a major
component of the desmosome in the upper epidermal layers, is associated with the pathogenesis of at least 3 skin
diseases: pemphigus foliaceus, staphylococcal scalded skin
syndrome, and autosomal dominant KPS.15
In the present study, we identified a heterozygous nonsense mutation, R26X, that causes a diffuse nonstriated
form of PPK. The R26X location at the start of the DSG1
coding sequence predicts loss of function of the mutant
allele due to either severe truncation or, more likely, nonsense-mediated messenger RNA decay. This mutation has
previously been described in a sporadic case of European ancestry affected with typical KPS.13 The variable
phenotypic expression of the mutation (diffuse PPK vs
KPS) in 2 cases and the reported absence of any phenotype in the mother of the proband suggest the influence
of epigenetic factors such as physical trauma to the pal(REPRINTED) ARCH DERMATOL/ VOL 141, MAY 2005
627
Figure 3. Sequence mutation analysis revealed in an affected individual a
heterozygous C→T transition (C76T; upper panel, arrow) resulting in the
substitution of a stop codon for an arginine residue at position 26 of the
amino acid sequence. The wild-type (WT) sequence (lower panel) is given for
comparison.
169 bp
139 bp
Figure 4. Findings of a polymerase chain reaction–restriction fragment
length polymorphism assay. To confirm segregation of R26X in the family, a
169–base pair (bp) fragment encompassing DSG1 exon 2 was amplified by
polymerase chain reaction and digested with BsiYI endonuclease. Since
R26X abolishes a recognition site for BsiYI endonuclease, healthy individuals
display a 139-bp fragment, while affected individuals display an additional
undigested 169-bp fragment.
moplantar skin. Interestingly, our proband denied any
physical activity for the past 5 years, possibly suggesting the existence of other epigenetic or genetic modifier
traits.
Despite the diffuse nature of the PPK affecting our
index case, histologic findings led us to focus our initial
molecular analysis on genes coding for desmosomal
components. It has been known for many years that, in
skin biopsy specimens obtained from patients with PPK,
epidermolytic changes in the upper epidermal layers
very often indicate mutations in KRT9 or KRT1.16 Our
data underscore the usefulness of another histopathologic clue for the diagnosis of PPK caused by mutations
in genes encoding desmosomal proteins, namely widening of the intercellular spaces and disadhesion of
suprabasal keratinocytes.
However, these histopathologic findings cannot be considered as entirely specific or sensitive for KPS; they must
be interpreted with caution because similar histologic features have been reported in other inherited desmosomal disorders,17,18 and these changes were absent in a
number of typical KPS cases caused by dominant mutations in DSG1 (including a patient carrying R26X).13 Thus,
to confirm our working diagnosis and restrict our subsequent mutation analysis, we developed a screening approach based on the use of 3 panels of microsatellite markWWW.ARCHDERMATOL.COM
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ers and haplotype analysis. While a formal mutation
analysis of all 3 genes involved in the pathogenesis of KPS
would have entailed the sequencing of more than 50 amplicons, by using haplotype analysis as a screening tool,
we identified DSG1 (comprising 15 exons only) as a target for subsequent mutation analysis. This integrative approach combining clinical ascertainment, pathogic examination, and candidate gene marker screening proved
to be efficient and led ultimately to the discovery of the
underlying genetic defect, despite the confusing phenotypic features displayed by the proband.
In summary, we have identified a recurrent mutation
in DSG1 that causes diffuse, and not striated, PPK. Our
data show that the diagnostic challenge posed by phenotypically heterogeneous PPK can be met through the
use of a comprehensive clinical, pathologic, and molecular approach.
Accepted for Publication: December 21, 2004.
Correspondence: Eli Sprecher, MD, PhD, Laboratory of
Molecular Dermatology, Department of Dermatology,
Rambam Medical Center, Haifa, Israel (e_sprecher
@rambam.health.gov.il).
Acknowledgment: We are grateful to all family members
for their participation in our study. We thank Vered Friedman, PhD, for outstanding DNA sequencing services.
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