From U385 INSERM, Faculté de Médecine, Avenue de
Valombrose, 06107 Nice cedex 2, France, the
§ Département d'Histologie, Embryologie, et
Cytogénétique, Faculté de Médecine
Lariboisière Saint Louis, 75730 Paris Cedex 15, France, the ¶ Institute of Molecular Medicine, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107-5541, and Service de Dermatologie, Hôpital Necker Enfants
Malades, 75730 Paris Cedex 15 Paris, France
Received for publication, January 16, 2001, and in revised form, March 13, 2001
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ABSTRACT |
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Laminin 5, the major keratinocyte adhesion
ligand, is found in the lamina lucida subregion of the epidermal
basement membrane of the skin, where it colocalizes with the anchoring
filaments. Mutations in the genes encoding laminin 5 cause junctional
epidermolysis bullosa, an inherited skin blistering disease
characterized by abnormal hemidesmosomes and cleavage of the
lamina lucida leading to epidermal detachment. In this work we describe
the genetic basis of a new subtype of lethal inherited epidermolysis
bullosa associated with reduced skin reactivity to laminin 5, presence of mature hemidesmosomes, and intradermal cleavage of the skin. The
epidermolysis bullosa patients were heterozygous for a nonsense mutation (Q896X) and a splice site mutation (764 Epidermolysis bullosa
(EB)1 is a heterogeneous
group of inherited diseases characterized by epithelial fragility to
mechanical friction resulting in blisters and erosions of the
integument. Extracutaneous involvement includes erosions of squamous
and transitional epithelia, pitted enamel, alopecia, and nail
dystrophia. The different forms of EB are associated with molecular
defects in components of the dermal-epidermal junction (1). The
dermal-epidermal junction is a complex basement membrane (BM)
ultrastructurally composed of four compartments: the ventral plasma
membrane of the basal keratinocytes and the associated hemidesmosomes;
the lamina lucida, which includes the anchoring filaments; the lamina densa; and the upper papillary dermis containing the anchoring fibrils
(2). About 20 biochemical components have so far been identified that
localize in the different compartments of the dermal-epidermal
junction. Among them type IV collagen, the backbone of all the basement
membranes, is the major component of the lamina densa; type VII
collagen is the prominent component of the anchoring fibrils; and
laminins 5, 6, and 10 localize both in the lamina lucida and the lamina
densa (3). The hemidesmosome-anchoring filament complex and the
anchoring fibrils form a specialized attachment structure common to the
stratified and simple epithelia exposed to the external environment
(3).
Based on ultrastructural data and antigen mapping, EB has been divided
into three major clinical subtypes according to the level of the
blister plane (4). In EB simplex, tissue separation occurs within the
basal keratinocytes as a result of genetic mutations in keratins 5 and
14 or in the hemidesmosomal component plectin. In junctional EB (JEB),
blisters arise within the lamina lucida, with the basal epidermal cells
at the roof of the blister and the lamina densa at its base. Tissue
cleavage results from mutations in the extracellular adhesion ligand
laminin 5 and the two transmembrane components of hemidesmosomes,
integrin Laminin 5 (the laminin It has been demonstrated that laminin 5 mediates epithelial cell
adhesion by interacting with integrins In this study, we have disclosed the molecular basis of an unusual form
of lethal epidermolysis bullosa associated with novel genetic mutations
in the LAMC2 gene. This form is characterized by a reduced
production of wild type laminin 5, the presence of mature
hemidesmosomes, and tissue separation underneath the lamina densa of
the dermal-epidermal junction. Our results provide the first evidence
that laminin 5 contributes to the firm adhesion of the epithelial
basement membrane to the underlying stroma.
Epidermolysis Bullosa Patients--
The two EB probands were
generated by healthy parents in a family with a history free of skin
diseases and genetic disorders. Proband 1 developed extensive cutaneous
blisters and erosions of the upper digestive tract shortly after birth.
EB was diagnosed on the basis of ultrastructural analysis of involved
skin that revealed severing of the dermal-epidermal junction. The
infant died at the age of 6 months. Proband 2 was the result of a
second pregnancy. At the 21st week of gestation, prenatal diagnosis
performed on fetal skin biopsies by electron microscope examination
detected epithelial detachment. The pregnancy was terminated.
Pathologic examination of the fetus was performed immediately after
delivery and confirmed the fragility of the skin and mucosa.
Biological Samples and Cells--
Primary keratinocytes isolated
from skin biopsies were cultured on lethally irradiated feeders of
mouse 3T3-J2 fibroblasts in the presence of a mixture (3:1) of DMEM and
Ham's F-12 medium (Life Technologies, Cergy-Pontoise, France). The
medium contained 10% fetal calf serum, 5 µg/ml insulin, 0.4 µg/ml
hydrocortisone, 0.1 nM cholera toxin, 10 ng/ml epidermal
growth factor, 2 nM triiodothyronine, and 0.18 mM adenine (27). Total RNA was purified from cultured keratinocytes using the RNableTM extraction kit (Eurobio, Les Ullis, France). Genomic DNA was extracted from peripheral blood following standard techniques.
Electron and Immunofluorescence Microscopy--
Ultrastructural
examination of noninvolved skin and mechanically induced blisters was
performed as described (28). Ultrathin sections were examined with a
JEOL EM 1200 transmission electron microscope. Indirect
immunofluorescence microscopy was performed on 5-µm sections of
frozen skin samples and on cultures of primary keratinocytes (28). The
samples were examined using a Zeiss Axiophot epifluorescence microscope.
Antibodies--
Antigen mapping was performed using monoclonal
antibody (mAb) GB3 raised against native laminin 5 (29), mAb K140
specific to the laminin Western Blot Analysis--
For Western blot analysis of the
extracellular matrix (ECM) deposited by the proband's keratinocytes,
subconfluent cell cultures were detached by treatment with 10 mM EDTA in phosphate-buffered saline (PBS). The ECM was
then scraped from the culture support in the presence of 2% SDS, 10%
glycerol, 100 mM Northern Blot Analysis--
30 µg of total RNA purified from
cultured keratinocytes was electrophoresed in a 1%
agarose-formaldehyde denaturing gel, transferred onto a nylon membrane
(Amersham Pharmacia Biotech), and hybridized with
32P-random-labeled laminin Detection and Verification of Mutations--
To search for
mutations in LAMC2 individual exons were PCR-amplified using
100 ng of genomic DNA as a template and oligonucleotide primer pairs
synthesized on the basis of the flanking intronic sequences (37).
Specifically, for amplification of the 230-bp DNA fragment comprising
the 189-bp exon 7 (nucleotides 764-953 in the cDNA;
GenBankTM number U31184) and containing the maternal
mutation 764 RT-PCR--
Total RNA isolated from the control and the
patient's cultured keratinocytes was reverse transcribed in a volume
of 25 µl using avian myeloblastosis virus reverse transcriptase as
recommended by the manufacturer (Promega, Madison, WI). One µl of
each reaction product was used to amplify overlapping regions of the
cDNAs spanning the open reading frame of the laminin
To isolate the wild-type RNA transcripts of the maternal
LAMC2 allele, 1 µl of the reverse transcription product
was submitted to allele-specific long range PCR amplification using the
Expand PCR system (Roche Molecular Biochemicals) to generate a
1.9-kilobase cDNA fragment (nucleotides 763-2705). The sense
primer (5'-CAAATTCTTGGGAATCAACAG-3') was homologous to the sequence
deleted in the maternal Quantification of Laminin 5 Deposited in the ECM by Keratinocyte
Cultures--
Laminin 5 deposition on plastic cell culture substrate
was quantified by enzyme-linked immunosorbent assay. Cells (2 × 103, 5 × 103, and 1 × 104/well) were seeded in multiwell plates (96 wells; TPP,
Zurich, Switzerland) and incubated for 48 h at 37 °C in a
humidified atmosphere in the presence of 5% CO2. The wells
were then washed twice in PBS and detached as previously described by
Delwel et al. (39). Briefly, cells were incubated overnight
at 4 °C in Hanks' buffered saline solution containing 20 mM Hepes, 20 mM EDTA, 1 mM EGTA, and a general protease inhibitor mixture (ICN Biomedicals, Aurora, OH)
and then dislodged by pipetting. The matrices were washed with PBS,
incubated for 10 min with PBS plus 1% Triton X-100 to remove cell
debris, and saturated for 1 h at 37 °C in PBS plus 2% bovine
serum albumin (BSA). Each well was incubated for 2 h at room
temperature with 50 µl of PBS plus 2% BSA containing mAb GB3 (10 µg/ml). The plates were washed three times with PBS and incubated
1 h at room temperature with 50 µl of PBS plus 2% BSA containing 1 µg/ml of anti-mouse HRP mAb (Dako) per well. After three
washes with PBS, 50 µl of a solution of o-phenylenediamine dihydrochloride (Sigma) were added to each well for 15 min in the dark
as devised by the supplier. The reaction was stopped by adding a 2 M solution of H2SO4, and color
yields were determined at 490 nm in an enzyme-linked immunosorbent
assay reader (Dynatech, Guyancourt, France).
Cell Detachment and Adhesion Assays--
Keratinocytes (3 × 104 cells/well) were plated in the presence of
serum-free medium in 96-well tissue culture plates (Polylabo, Strasbourg, France). The wells had previously been coated with 0.5%
bovine serum albumin fraction V (Sigma) or 10 µg/ml Engelbreth Holm-Swarm Mouse Sarcoma matrigel (Sigma) by overnight adsorption at
4 °C, washed twice with PBS, and saturated for 1 h at room temperature with 0.5% heat-inactivated BSA in PBS. Cells were allowed
to adhere for 1, 16, or 24 h at 37 °C. After washing with PBS,
the attached cells were fixed with 3% paraformaldehyde for 20 min at
room temperature, washed three times with PBS, and stained with 0.5%
crystal violet in 20% methanol for 15 min. Excess dye was washed off
with water, and the stained cells were eluted with 100 µl of a
solution containing 50% ethanol and 0.1 M sodium citrate, pH 4.2. Absorbance at 560 nm was read in a microplate manager (Bio-Rad). Nonspecific adhesion was evaluated by seeding the
keratinocytes in wells coated with 0.5% BSA. Each assay point was
derived in triplicate. For cell detachment assays, suspensions of
secondary keratinocytes (6 × 104
cells/cm2) were seeded in tissue culture flasks and
incubated for 48 h at 37 °C. The cell cultures were then rinsed
and treated with a solution of 0.05% trypsin, 0.01% EDTA (0.5 M), pH 8. The number of cells detached at different times
was determined by collecting the supernatants of each cell culture and
direct cell counting.
All measurements were performed in triplicate, and the results obtained
from the three different experiments were expressed as the mean of
S.D.
The Intradermal Cleavage of the Dermal-Epidermal Junction Is
Associated with a Reduced Immunoreactivity of Laminin 5--
The
probands presented with the characteristic clinical phenotype of severe
EB. Ultrastructural examination of nonlesional skin detected mature
hemidesmosomes and well formed anchoring filaments connected to the
lamina densa of the basement membrane zone (Fig.
1A). In fresh lesions of
blistered skin, the cleavage plane localized below the lamina densa,
and hemidesmosomes were well formed (Fig. 1B). Careful
examination of the involved skin also detected lesions with the cleft
lying within the lamina lucida and presenting normally shaped
hemidesmosomes at the roof of the blister (Fig. 1C).
Immunoreactivity of noninvolved skin to mAb GB3 against laminin 5 was
strongly reduced compared with the control skin of nonaffected donors
(Fig. 2, A and B).
In contrast, immunoreactivity of integrin The EB Probands Are Compound Heterozygous for Mutations in the
LAMC2 Gene--
The finding that the abnormal expression of laminin 5 is associated with the presence of well formed hemidesmosomes and a cleavage plane of the skin lying below the lamina densa is intriguing, because so far the altered expression of laminin 5 has always been
associated with tissue separation within the lamina lucida and
dysplastic hemidesmosomes (40, 41). To confirm the direct implication
of laminin 5 in the condition, the expression level of the laminin
To identify possible pathogenic genetic mutations in LAMC2,
the probands' genomic DNA was PCR-amplified using primers homologous to the intronic DNA sequences flanking each exon of LAMC2
(37). Conformation-sensitive gel electrophoresis revealed heteroduplex formation with the PCR products corresponding to exon 18 (not shown).
Direct nucleotide sequencing of the amplimers detected a heterozygous
C
Heteroduplex formation was also observed with the PCR products
encompassing exon 7. Direct nucleotide sequencing of the PCR products
detected an intronic T The EB Keratinocytes Express Wild-type Laminin 5--
Similar to
the probands' epidermis, the probands' keratinocytes expanded in
culture were weakly reactive to pAb SE144 (Fig. 6A) and to mAb GB3 (data not
shown). Accordingly, the ECM deposited by these cells on the culture
vessel was faintly stained compared with the ECM layered down by
wild-type keratinocytes, indicating deposit of trimeric laminin 5. Expression of the
In light of these results, we hypothesized that the maternal mutation
764 The Proband's Keratinocytes Display Reduced Adhesion
Capacity in Vitro--
Secretion of laminin 5 accounts for the
assembly of mature hemidesmosomes in the patients' skin and promotes
cell adhesion both in vivo and in vitro (42). To
evaluate the adhesive capacity of the probands, keratinocyte cell
suspensions were seeded either on EHS matrigel substrate or on plastic
coated with BSA. Secondary
Because in the absence of the ECM culture substrate the adhesion of the
proband's keratinocyte was comparable with that of the
Taken together, these observations underscore the dramatic adhesion
deficiency of the proband's keratinocytes but also indicate that
production of scant amounts of laminin 5 plays a perceptible effect on
cell adhesion that could explain the sublamina densa cleavage detected
in the two EB patients.
In this study, we have examined two cases of a rare variant of
inherited lethal EB. Blistering was associated with a reduced expression of laminin 5, as illustrated by the barely detectable immunoreactivity of the probands' skin to the monoclonal antibody GB3,
which recognizes native laminin 5, and to the polyclonal antibody SE144
directed against the laminin The two EB patients were found to be compound heterozygotes for two
novel mutations in the LAMC2 gene. As attested by Northern blot analysis of total RNA purified from one of the probands' keratinocytes, the genetic defects led to reduced levels of The other mutation was a single base pair substitution (764 Intronic mutations adjacent to the 5' splice site reduce the efficiency
of mRNA splicing and activate cryptic splice sites (47). Legitimate
splicing may, however, take place at a reduced rate, provided that
spliceosome assembly is not abolished (48). In our patients,
allele-specific RT-PCR amplification of the mRNA demonstrated that
a wild-type Laminin 5 adheres efficiently to surfaces and primes the plastic and
glass culture vessels for cell adhesion (50-52). Lines of evidence
have indicated that accumulation of the extracellular matrix ligand at
a threshold level is required for efficient spreading and cell adhesion
(42, 53). The amount of In conclusion, our observations expand the spectrum of genetic
heterogeneity in EB and also demonstrate the critical contribution of
laminin 5 to the cohesion of the epithelial basement membrane. Furthermore, they underscore the need for a correct evaluation of the
effect associated with the transfer of curative genes in EB. Phenotypic
reversion of keratinocytes defective for proteins of the
hemidesmosome-anchoring fibril complex has recently led to proposals
for gene therapy of inherited EB (55). In this context and according to
observations made on transgenic mice (56), here we show that assembly
of ultrastructurally normal hemidesmosomes may not reflect a full
recovery of cell adhesion but only partial rescue due to insufficient
expression of the curative polypeptide.
10T
G) in the gene
(LAMC2) for the
2 chain of laminin 5. The
nonsense mutation causes accelerated decay of the corresponding
mRNA, while the splice site mutation results in maturation of a
cryptic wild-type
2 mRNA leading to reduced expression of
wild-type laminin 5. In vitro studies using the
probands' keratinocytes showed that secretion of reduced
amounts of functional laminin 5 in the patient, although permitting
formation of hemidesmosomes, fail to restore efficient cell
adhesion. Our results provide the first evidence that laminin 5 contributes to the firm adhesion of the epithelial basement membrane to
the underlying stroma. They also show that a low expression level of
laminin 5 induces assembly of mature hemidesmosomes in vivo
but fails to assure a stable cohesion of the dermal-epidermal junction.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
4 and collagen type XVII. In
dystrophic EB, the split localizes within the papillary dermis below
the lamina densa and results from mutations in collagen type VII.
Immunostaining of EB skin using an adequate panel of antibodies may
detect the abnormal expression of a specific BM component and identify
the candidate gene(s) in the disease (4).
3
3
2 heterotrimer) is specifically
associated with the hemidesmosome-anchoring filament complexes (5, 6).
It has been reported to influence morphogenetic events (7, 8), to
affect cell motility (9, 10), to enhance invasiveness in human cancers
(11-15), and to control cell growth (16). In the skin, laminin 5 is
synthesized and secreted into the extracellular matrix by the basal
keratinocytes and constitutes a specific substrate for static
attachment and for adhesion of proliferating and migrating cells. Null
mutations in any of the three genes (LAMA3,
LAMB3, and LAMC2) encoding laminin 5 cause the
extensive epithelial disadhesion distinctive of lethal JEB (H-JEB, for
Herlitz JEB) (17). Mutations leading to synthesis of an aberrant but
partially functional laminin 5 result in mild JEB (nH-JEB, for
non-Herlitz JEB), which is characterized by a limited tendency to skin
blistering. In H-JEB, hemidesmosomes are absent or hypoplastic and/or
reduced in number. In nH-JEB, hemidesmosomes may be reduced in number,
lack the hemidesmosomal subbasal dense plate, and have rudimentary
cytoplasmic plaques. Transfer of a curative transgene that rescues
expression of functional laminin 5 in H-JEB keratinocytes restores
hemidesmosome assembly and cell adhesion (18).
6
4
and
3
1 through its COOH-terminal G-domain
(19-21). In blistered nH-JEB skin, immunoreactive laminin 5 distributes along the floor of the blister, which indicates interaction
of the protein with components of the lamina densa and the papillary
dermis. Furthermore, laminin 5 binds the NC-1 domain of collagen type
VII (22, 23) and associates with laminin 6 in the skin and laminin 7 in
the amnion (24, 25). Additional evidence supporting a structural
function of laminin 5 in basement membrane stability includes
immunochemical data, which suggests that initiation of skin basement
membrane formation involves assembly of laminin 5 into laminin
complexes and interaction with integrins
1 and
4 (26). However, the active role of laminin 5 in
structuring of the basement membrane has not been substantiated by
direct in vivo observations and remains hypothetical.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 chain (30), and polyclonal antibodies
(pAbs) SE85 and SE144 specific to the laminin
3 and
2 chains,
respectively (31, 32). mAb GoH3 is specific to integrin
6 (33). A pAb anti-laminin 1 (L9393; Sigma), a pAb
anti-collagen type IV (10411; Institut Pasteur, Paris, France), and the
pAb LH7:2 directed against collagen type VII (34) were also employed.
-mercaptoethanol, 50 mM
Tris-HCl, pH 6.8. The samples were briefly sonicated and cleared by
centrifugation at 10,000 × g for 5 min at 4 °C. The protein concentration was determined by the Bradford method (Bio-Rad). 25 µl of the ECM and serial dilutions of the original solution in PBS
were electrophoresed in a 6% acrylamide SDS-polyacrylamide gel and
blotted on nylon filters (35). The antibody-antigen complex was
revealed using the ECL Western blotting kit (Amersham Pharmacia
Biotech). Quantitation of autoradiograms was performed by densitometric
scanning using the Bioprofil software program (Vilber Lourmat, France).
3,
2, and
glyceraldehyde-3-phosphate dehydrogenase cDNA probes as described
(36).
10T
G, the following primers were used:
5'-TCATAGCCCAAAGTGCATA-3' (forward) and
5'-AGGCTTGTTGAACAAATGAA-3' (reverse) (37). For amplification of the
152-bp exon 18 (nucleotides 2601-2753 in the cDNA;
GenBankTM number U31195) containing the paternal mutation
Q896X, the primers were 5'-GGTGTGGTAACTGGTAAGCA-3' (forward) and
5'-TATTCATATGGTGGGCATTC-3' (reverse). The PCR conditions were as
follows: 95 °C for 5 min followed by 30 cycles at 94 °C for
30 s, 58 °C for 45 s, and 72 °C for 30 s. 5 µl
of the PCR products were used for heteroduplex analysis by
conformation-sensitive gel electrophoresis (38) followed by direct
automated sequencing of the PCR products containing a heteroduplex band
(ABI, Foster City, California). The presence of mutations Q896X and
764
10T
G in members of the family and unrelated healthy controls
was assessed by direct sequence analysis. Occurrence of mutation
764
10T
G was further investigated in 100 alleles of unrelated
healthy controls by allele-specific oligonucleotide hybridization as
described (36). The allele-specific oligonucleotides were
5'-GCTGTGAAACAGAGTTTTA-3' and
5'-GCTGTGAAACCGAGTTTTA-3', corresponding to the wild type
and mutant LAMC2 sequences, respectively.
2 chain.
Specifically, for amplification of the 439-bp cDNA fragment
(nucleotides 588-1027; GenBankTM number X73902) (31)
including the 23-bp deletion generated by the maternal mutation
764
10T
G, the oligonucleotide primers were
5'-GCAGCTTCTGCAGAATACAGT-3' (forward) and 5'-AGATTCCGCAGTAACCTTCG-3' (reverse). The PCR conditions were as follows: 94 °C for 3 min followed by 30 cycles at 94 °C for 20 s, 58 °C for 45 s, and 72 °C for 45 s. The amplification products were
submitted to direct automated sequencing.
2 cDNA bearing the mutation
764
10T
G. The antisense primer (5'-CCAGTTTCCCAGATTCTTCTG-3', nt
2685-2705) was homologous to the maternal
2 cDNA fragment harboring guanine 2688 (which in the paternal
2 cDNA is
substituted by a polymorphic adenosine). The PCR conditions were as
follows: 94 °C for 3 min, 94 °C for 20 s, 52 °C for
45 s, 68 °C for 1 min 30 s (10 cycles), 94 °C for
20 s, 52 °C for 45 s, and 68 °C for 2 min (25 cycles
with an increment of 20 s in extension time at each stage). Direct
sequence analysis of the amplimer was performed using the primer
5'-GATTCAGTGTCTCCGCTTCA-3' (nucleotides 2557-2576).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 (Fig. 2,
G and H) and that of all of the major basement
membrane components (not shown) was comparable with that detected in
control skin. This finding suggested the possibility of a potential
association of the disease with an altered expression of laminin 5. Indeed, labeling with the anti-laminin
2 pAb SE144 was faint (Fig.
2, C and D), while staining with mAb K140 (Fig. 2, E and F) and pAb SE85 (data not shown)
specific to the laminin
3 and
3 chains, respectively, was
slightly decreased. These observations suggested that LAMC2
was the candidate gene in the condition. In the lesional skin,
immunostaining of laminin 5, type IV collagen, laminin 1, and type VII
collagen was located at the roof of the blisters (Fig.
3, A-D), which confirmed the sublamina densa severing of the basement membrane. In a few blisters, the cleavage plane partially localized within the lamina lucida, above
the lamina densa (Fig. 3, insets).
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Fig. 1.
Electron microscopy examination of the
proband's skin. A, low magnification of the basement
membrane zone in nonlesional skin reveals normal ultrastructural
features. The numerous hemidesmosomes (HD) contain well
defined inner (ip) and outer (op) plaques and are
associated with bundles of keratin intermediate filaments
(IF). In the lamina lucida (LL), the subbasal
dense plates of the hemidesmosome are connected with the anchoring
filaments (Af) abutting on the lamina densa (LD),
opposite to anchoring fibrils (AF). B, in
blistered skin, the cleavage plane is prevalently located beneath the
lamina densa. C, in blisters presenting a cleft plane
(asterisk) within the lamina lucida, the
hemidesmosome-anchoring filament complexes are at the roof of blisters
and connected with keratin intermediate filaments. Scale
bars, 0.5 µm.
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Fig. 2.
Immunohistochemical analysis of the
noninvolved skin from proband 1. In cryostat skin sections,
staining of the basement membrane using mAb GB3 specific to native
laminin 5 was attenuated and discontinuous (A) compared with
healthy controls (B). Immunoreactivity of each individual
chain of laminin 5 is also reduced with a strong attenuation of the
fluorescent signal with the anti- 2 pAb SE144 (C) and a
less marked reduction with the anti-
3 mAb K140 (E) and
anti-
3 pAb SE85 (not shown). In contrast, immunoreactivity to mAb
GoH3 specific to the integrin
6 subunit is comparable in
the patient (G) and in the control (H).
Scale bar, 48 µm.
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Fig. 3.
Immunohistochemical analysis of the lesional
skin in proband 2. Immunostaining to laminin 5 (A)
collagen type IV (B), laminin 1 (C), and collagen
type VII (D) is localized at the roof of the blisters,
indicating that the cleavage plane of the basement membrane zone lies
beneath the lamina densa. In a subungula blister, the basement membrane
components localized at the floor of the lesion, indicating that the
sewerage localizes within the lamina lucida (insets).
Scale bars, 48 µm.
3
and
2 chains was determined by Northern blot analysis of total RNA
purified from keratinocytes isolated from the EB fetus. Compared with
control keratinocytes, expression of the laminin
2 chain was 80%
reduced in the patient's cells, whereas the steady state level of the
laminin
3 transcripts was not affected (Fig.
4).
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Fig. 4.
Northern analysis of the patient's
keratinocytes. Northern blot analysis was performed using total
RNA purified from primary cultures of keratinocytes obtained from the
proband (P) and an unrelated control (C). The RNA
was electrophoresed on denaturing agarose gels, blotted to a
nitrocellulose sheet, and successively probed with
32P-labeled cDNAs for the laminin 3 and laminin
2
chains. A reduction in the steady-state level of the
2 messenger RNA
was noted in the proband's keratinocytes.
T transition at position 2686 of the laminin
2 cDNA
(GenBankTM accession number X73902), which substitutes a
termination codon TAA for glutamine 896 (CAG) (Fig.
5A). The mutation was designated Q896X. A conservative G
A transition was also detected at
the wobble position of the codon for glutamine 896 (Fig.
5A). Direct nucleotide sequencing of the PCR products
amplified from the genomic DNA of the proband's parents showed that
mutation Q896X and polymorphism 2688 G
A co-segregate with the
paternal allele (not shown).
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Fig. 5.
Identification of the genetic mutations in
LAMC2. A, paternal mutation.
Direct nucleotide sequencing of the proband's genomic DNA
(lower panel) revealed a heterozygous C T
transition at nucleotide position 2686. This 2686 C
T nucleotide
change substitutes a glutamine codon (CAG) with a stop codon (TAA) at
amino acid position 896. The mutation was designated Q896X. In the
mutated allele, a polymorphic G
A transition was also detected at the
wobble position (nt 2688) of the codon for glutamine 896 (asterisk). The presence of mutation Q896X in the EB kindred
was assessed by nucleotide sequencing of the genomic DNA obtained from
the patients' relatives. The mutated allele was detected in the
probands' father and in the affected fetus. WT, wild type.
B, maternal mutation. Direct nucleotide sequencing of the
proband 230-bp PCR amplimer, spanning exon 7 of LAMC2 and
its intronic boundaries, detected a heterozygous T
G transversion at
position
10 of intron 6 as shown by the sequence of the reverse
strand (lower panel). The mutation was designated
764
10T
G. Direct sequencing of the genomic DNA from the mother
detected mutation 764
10T
G in one allele. C, direct
nucleotide sequencing of the cDNA reverse-transcribed from the
laminin
2 of the patient identified a heterozygous out of frame
23-bp deletion (nucleotides 764-787 of the
2 cDNA) as shown by
the sequence of the reverse strand. D, schematic
representation of the aberrant splicing of the
2 pre-mRNAs
transcribed from the maternal LAMC2 allele. Mutation
764
10T
G activates a cryptic splice site (AG, nucleotides 785 and
786 in the
2 cDNA sequence) within exon 7 and induces an
out-of-frame internal deletion of 23 nucleotides.
G transversion at position
10 of exon 7 (764
10T
G) in the patient (Fig. 5B), in the affected fetus, and in the mother. To determine the effect of mutation 764
10T
G at the mRNA level, the laminin
2 cDNA was
amplified by RT-PCR using total RNA purified from the proband's
keratinocytes. Direct nucleotide sequencing of the 439-bp cDNA
fragment encompassing exons 6 and 7 of LAMC2 identified a
wild type mRNA and an internally shortened transcript bearing a
23-nt out-of-frame deletion at the 5'-end of exon 7 (nt 764-786 of the
laminin
2 cDNA). This deletion results in a shift of the open
reading frame and makes a downstream premature termination codon
(TGA at nt 813-816; Fig. 5C). The internally deleted
mRNA is generated by an abnormal splicing involving a putative
cryptic acceptor splice site within exon 7 (cag/G, nt 784-787) (Fig.
5D). The possibility that mutation 764
10T
G constitutes
a polymorphic variation in the population was excluded by
allele-specific oligonucleotide screening of a panel of 100 unrelated
control alleles (data not shown). Screening for mutations detected no
additional base change either in the remaining LAMC2
cDNA sequences or introns 5-7. We therefore concluded that
mutations Q896X and 764
10T
G are causative for EB phenotype in the
kindred. This conclusion was confirmed by the results of an early
DNA-based prenatal testing performed in a subsequent pregnancy.
Genotyping of the fetal genomic DNA prepared from a chorionic villous
biopsy obtained at 10 weeks of pregnancy showed that the fetus had
inherited the wild type LAMC2 alleles. At delivery, the
newborn was clinically unaffected.
2 polypeptide by the proband's keratinocytes was
assessed and quantified by immunoblotting of the ECM deposited by these
cells on the Petri dishes using pAb SE144. Two hybridization bands were
detected with the apparent electrophoretic mobility of the
unprocessed (155 kDa) and extracellularly processed (105 kDa)
2
polypeptides (Fig. 6B) (25). By densitometric analysis, the
intensity of the two bands was estimated to be 100-fold weaker than
that of the corresponding bands detected in cultures of wild type
keratinocytes.
View larger version (69K):
[in a new window]
Fig. 6.
Patient's keratinocytes secrete wild type
laminin 5. A, indirect immunofluorescence analysis of
keratinocyte cultures obtained from the proband (a) and
labeled using pAb SE144 show staining of the cytoplasm and the ECM.
a was overexposed 2× with respect to keratinocyte cultures
obtained from a healthy control (b). The H-JEB keratinocytes
are not reactive to the antibody (c). B, Western
blot analysis of the ECM secreted in culture by the patient
(P) and control (C) keratinocytes. The ECM
proteins were fractionated on a denaturing SDS-6% polyacrylamide gel,
blotted onto a nitrocellulose filter, and reacted with pAb SE144
specific to laminin 2 chain. The intensity of the bands
corresponding to the extracellular forms (155 and 105 kDa) of the
laminin
2 chain was estimated by comparison with serially diluted
samples of ECM obtained from control keratinocyte cell cultures
(lane 1, 1:100 dilution; lane
2, 1:50; lane 3, 1:20; lane
4, 1:10; lane 5, 1:5; lane
6, undiluted). Molecular weight markers are indicated on the
left. C, quantification of laminin 5 layered down
to the culture substrate by the keratinocyte cultures. Cells were
seeded in multiwell plates, allowed to adhere for 48 h, and then
processed as detailed under "Materials and Methods." Quantification
of native laminin 5 (
3
3
2) deposited on the culture substrate
was determined by enzyme-linked immunosorbent assay using mAb GB3.
Values are the mean of quadruplicates ± S.D. Keratinocytes
isolated from a healthy control (black bar),
proband 2 (shaded bar), and a
2-null JEB
patient (white bar). D, specific
amplification by long range RT-PCR of the RNA transcripts encoded by
the maternal LAMC2 allele of the proband (P)
detected expression of a wild-type
2 mRNA (arrow).
M, molecular weight markers; C, control without
cDNA.
10T
G permits synthesis of full-length
2 polypeptides that
incorporate into laminin 5 heterotrimers. To verify this possibility,
incorporation of native laminin 5 in the extracellular matrix layered
down by the proband's keratinocytes was determined by enzyme-linked
immunosorbent assay using mAb GB3. As shown in Fig. 6C, wild
type keratinocytes efficiently deposited laminin 5 on the plastic
culture substrate, while the proband's keratinocytes secreted barely
detectable amounts of the protein. Taking advantage of the polymorphic
nucleotide 2688 detected in the paternal LAMC2 allele,
synthesis of a wild-type
2 mRNA was then verified by long range
allele-specific RT-PCR amplification of the
2 RNA transcripts using
total RNA purified from the probands' keratinocytes as a template. The
antisense primer was specific to the maternal
2 cDNA fragment
spanning nt 2686-2705, and the sense primer was complementary to the
23-bp cDNA sequence absent in the internally deleted maternal
2
mRNA. Direct sequencing showed that the PCR amplification products
(nt 763-2705 of the
2 cDNA) contained the guanine residue
encoded at nt 2688 by the maternal LAMC2 allele and
harbored the 23-bp sequence previously found to be deleted in the
maternal transcripts. These results demonstrated that the maternal
mutation 764
10T
G allows rescue of a minor form of wild type
2
pre-mRNA (Fig. 6D).
2-null keratinocytes isolated from
an unrelated H-JEB (43) and wild type keratinocytes were used as
controls. Similar to the normal keratinocytes, the number of the
proband's cells sticking to the EHS matrigel substrate increased with
time and at a comparable extent, while the percentage of adhering
2-null keratinocytes increased much slower. In the absence of
matrigel, only the wild type keratinocytes efficiently attached to the
culture substrate, indicating that the extracellular matrix layered
down by the EB keratinocytes was inefficient in promoting cell adhesion
on unprimed plastic culture substrate (Fig.
7A).
View larger version (13K):
[in a new window]
Fig. 7.
Adhesion and detachment kinetics of
keratinocyte cell cultures obtained from proband 2. A,
cells (3 × 105 cells/well) were plated in
96-microtiter well plates coated with BSA (0.5%) or EHS matrigel (10 µg/ml). After 1-, 16-, and 24-h incubation at 37 °C, the cells
were fixed and stained, and their number was evaluated as described
under "Materials and Methods." Values are the mean of
triplicates ± S.D. cells from a healthy control (black
bar), proband 2 (shaded bar), and a
2-null JEB patient (white bar). B,
detachment kinetics of subconfluent keratinocyte cell cultures obtained
from proband 2 (black circle), a healthy control
(black square), and an unrelated H-JEB patient
(white circle) in the presence of trypsin. The
number of cells detached at the indicated time of trypsinization was
determined by direct cell counting. Each point is the average of
triplicates from three independent experiments and represents the
mean ± S.D. of the dislodged cells. Variation among triplicates
was <5%.
2-null
keratinocytes, the adhesive capacity of these cells was further
assessed using a cell detachment kinetic assay (22). Exponentially
growing cell cultures were treated with a trypsin/EDTA solution, and
the number of cells dislodged at increasing intervals of time was
evaluated by direct counting. The results showed that the proband's
keratinocytes rapidly detached from the cell culture support. However,
these keratinocytes displayed a higher adhesion capacity than the
2-null counterparts because only 12% of them were dislodged after 6 min and 47% after 8 min, while for the
2-null cells, values were 52 and 83%, respectively (Fig. 7B). At 15 min, however, 100%
of the EB keratinocytes were detached compared with 25% of the wild
type cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 chain. Previously, lethal EB caused
by hampered expression of laminin 5 has been associated with the
assembly of sparse and/or incomplete hemidesmosomes, resulting in a
pronounced tendency to dermal-epidermal tissue separation in the plane
of the lamina lucida of the basement membrane zone. In these two
patients, transmission electron microscopy detected normally formed
hemidesmosomes, and blister formation predominantly localized beneath
the lamina densa. Tissue separation at the basement membrane/dermis
interface was confirmed by immunohistochemical staining of the lesional
skin that mapped all of the major components of the basement membrane
to the roof of the blisters. It is unlikely that artifacts caused the
atypical cleavage of the basement membrane, because the ultrastructural
and immunohistochemical analyses were performed using fresh
mechanically induced blisters, and skin biopsy specimens were processed
following well established procedures routinely used to study EB
epidermis. The presence of structurally normal hemidesmosomes in a
patient suffering from severe junctional EB has been previously
reported in only one case (41). The patient died a few days after
birth, and neither immunomapping of the dermal-epidermal junction nor
genetic studies were performed. Therefore, here we present the first
evidence that in patients with lethal EB, laminin 5 mutations may
permit assembly of structurally normal hemidesmosomes and cause
blisters with a sublamina densa cleft of the dermal-epidermal junction.
2 mRNA expression. One of the mutations was a nonsense mutation (Q896X) within the
-helical coiled-coil domain of the polypeptide, which predicted a truncation of the laminin
2 chain by 75%. It has
been demonstrated that premature termination codons cause accelerated
decay of the abnormal RNA transcripts and absent expression of the
corresponding polypeptide (44). The shortened
2 polypeptide is not
expected to incorporate into laminin 5, because the COOH-terminal coiled-coil domain I of the long arm that is crucial for assembly of
the laminin heterotrimers is missing (Refs. 45 and 46 and references
therein). In addition, Western blot analysis with keratinocyte protein
extracts using the polyclonal antibody SE144 (31) was negative for the
presence of the shortened
2 polypeptide with an expected size of
~100 kDa. It is therefore likely that the mRNA transcript
containing the nonsense mutation undergoes rapid decay, which prevents
synthesis of the aberrant polypeptide.
10T
G)
at the 3' splice site of intron 6 that activates a putative cryptic
splice site (CAGG) within exon 7 on the maternal
LAMC2 allele. The aberrant splicing generates a mutated
mRNA with an internal out-of-frame deletion that results in a
downstream premature termination codon predicting truncation within the
NH2 terminus (domain IV) of the
2 chain short arm.
Absent synthesis of the aberrant polypeptide due to mRNA decay is
also likely.
2 transcript is synthesized from the maternal allele
containing mutation 764
10T
G. According to these results and the
faint immunoreactivity of skin and cultured keratinocytes to monoclonal
antibody GB3, Western blot analysis of the extracellular matrix
deposited by the proband's keratinocytes in a culture vessel detected
laminin 5 molecules with a full-length
2 polypeptide. Rescue of wild
type transcripts from pre-mRNA with intronic mutations has been
documented previously (47). In particular, moderation of the skin
blistering in two unrelated EB patients has been associated with
intronic mutations in the genes COL7A1 and ITGB4,
which allow expression of normal collagen type VII and integrin
4 polypeptides, respectively (36, 49). In our patients,
expression of cryptic wild type
2 mRNA restored secretion of
functional laminin 5 molecules that mediated assembly of
ultrastructurally normal hemidesmosomes but did not support epithelial adhesion.
2 chain detected in the ECM produced
in vitro by the proband's keratinocytes was 100-fold lower
than in normal keratinocytes, and the
2 polypeptides were
efficiently processed as judged from the respective intensity of the
155- and the 105-kDa bands visualized by Western blot analysis. The
highly restricted production of
2 chain results in secretion of
laminin 5-containing extracellular matrix unable to support stable
adhesion. Indeed, adhesion assays showed that the probands' keratinocytes adhere poorly to plastic coated with BSA, while they
attach efficiently to plastic precoated with EHS matrigel. This
observation underscores the inability of scant amounts of laminin 5 to
form a high affinity adhesion substrate in vitro and also
provides a possible explanation for the unusual ultrastructural features of the patients' skin. Indeed, it has been shown that in
healthy skin 90% of laminin 5 concentrates immediately under the
hemidesmosomes, while the remaining 10% distributes along the basement
membrane, where it is thought to promote stable epithelial-stromal attachment by interacting with laminin 6/7 (5, 24, 54). In our
patients, scant expression of laminin 5 in the basement membrane
apparently may favor its accumulation on top of the anchoring fibrils,
which mediates efficient nucleation of hemidesmosomes, and limit its
deposition in the interhemidesmosomal space and in the anchoring
plaques of the papillary dermis (23). The sub-lamina densa cleavage of
the blistered skin may thus reflect both the firm fastening of the
anchoring fibrils to the lamina densa and the fragility of the deepest
layers of the dermal-epidermal junction. On the other hand, the
junctional cleavage noted in a number of blisters may result from
insufficient deposition of laminin 5 underneath the hemidesmosome,
which weakens the connection between the anchoring filaments and the
lamina densa, as observed in JEB patients with mutations causing loss
or synthesis of abnormal laminin 5.
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ACKNOWLEDGEMENTS |
---|
We acknowledge A. Spadafora, M. Heller, and J-P. Monnet for technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from EEC BIOMED 2 (BMH4-97-2062), the Program Hospitalier de Recherche Clinique (France), the DEBRA Foundation (United Kingdom), and the Association Francaise contre les Myopathies (France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The first two authors contributed equally to this paper.
** To whom correspondence should be addressed: INSERM U385, U.F.R de Médecine, Av. de Valombrose, 06107 Nice cedex 2, France. Tel.: 33 493 37 77 79; Fax: 33 493 81 14 04; E-mail: meneguzz@unice.fr.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M100381200
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ABBREVIATIONS |
---|
The abbreviations used are: EB, epidermolysis bullosa; BM, basement membrane; JEB, junctional EB; mAb, monoclonal antibody; pAb, polyclonal antibody; ECM, extracellular matrix; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; nt, nucleotide(s); BSA, bovine serum albumin.
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