Download The anhidrotic ectodermal dysplasia gene (EDA) undergoes

 1998 Oxford University Press
Human Molecular Genetics, 1998, Vol. 7, No. 11 1661–1669
ARTICLE
The anhidrotic ectodermal dysplasia gene (EDA)
undergoes alternative splicing and encodes
ectodysplasin-A with deletion mutations in
collagenous repeats
Mònica Bayés, Andrew J. Hartung1, Sini Ezer, Johanna Pispa2, Irma Thesleff2,
Anand K. Srivastava1,* and Juha Kere*
Department of Medical Genetics, Haartman Institute, PO Box 21, University of Helsinki, 00014 Helsinki, Finland,
1J. C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC 29646, USA and
2Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Received April 27, 1998; Revised and Accepted July 10, 1998
GenBank accession nos AF040628, AF061189–AF061194, AF060777–AF060788
Anhidrotic ectodermal dysplasia (EDA) is an X-linked recessive disorder which affects ectodermal structures. A
cDNA encoding a 135 amino acid protein with mutations in 5–10% of EDA patients has been reported. We have
built up a complete splicing map of the EDA gene and characterized the longest and what most probably
represents the full-length EDA transcript, EDA-A. It encodes a 391 amino acid transmembrane protein with a short
collagenous domain, (Gly-X-Y)19, and is highly homologous to the protein mutated in Tabby mice (Ta-A). Four new
transcripts that code for truncated proteins lacking the collagenous domain were also detected. The splice
variants show different expression patterns in eight tissues analyzed, suggesting a regulatory mechanism for
gene expression. The EDA-A form of the protein is transported to the cell membrane and induces rounding of the
cells, properties also associated with the 135 amino acid isoform. We have determined the genomic organization
and the exon–intron boundaries of the EDA gene. SSCP analysis of the nine exons corresponding to EDA-A
allowed the identification of mutations in 12 out of 15 EDA patients. Interestingly, three mutations removed either
two or four of the Gly-X-Y repeats without interrupting the reading frame, thus suggesting a functional role for
the collagenous domain. Our results will allow mutation diagnostics in the majority of patients.
INTRODUCTION
Hypohidrotic or anhidrotic ectodermal dysplasia (EDA) is an
X-linked recessive disorder characterized by the absence or
hypoplasia of hair, teeth and sweat glands (MIM 305100) (1). The
trait had been mapped to Xq12–q13 and the gene responsible for
the disease was isolated by positional cloning (2). In that first
report an EDA cDNA encoding a 135 amino acid protein
(referred to in this paper as EDA-O, for original) was described.
Structure predictions suggested that the EDA polypeptide might
belong to the class II transmembrane proteins and its membrane
localization has been confirmed (3). Northern and in situ
hybridization experiments revealed that the gene is expressed in
the skin and its appendages, but also in other tissues and organs
not affected in EDA patients (2,4). Despite an extensive search
for mutations in the EDA-O coding sequence, the molecular
defect has been identified in only 5–10% of EDA patients (2,5).
An extended search for additional transcripts recently resulted in
the isolation of an alternatively spliced mRNA, coding for a
protein of 178 amino acids with different C-terminal residues
(EDA-B) (6).
The Tabby (Ta) phenotype in mice resembles that of EDA
patients and it has been suggested to represent mutation of an
orthologous gene. Indeed, a highly similar murine gene was
recently isolated and mutations in the gene were found in Ta mice
(6,7). Altogether, four different Ta mRNAs, most likely arising by
alternative splicing, have been reported. Ta-A codes for the longest
predicted protein (391 amino acids), which contains an additional
stretch of 19 Gly-X-Y repeats, a putative glycosylation site and a
cysteine-rich C-terminal region. Ta-B (177 amino acids) is the
mouse counterpart of EDA-B, while in Ta-C (220 amino acids)
part of the intron 1 sequence is retained and transcribed (6). In
addition, a variant of Ta-A with 378 amino acids has been reported
(7). The protein family was named ectodysplasin-A (6) and may
*To whom correspondence should be addressed. J.K.: Tel: +358 9 1912 6538; Fax: +358 9 1912 6677; Email: [email protected]; A.K.S.: Tel: +1 864 388
1806; Fax: +1 864 388 1808; Email: [email protected]
1662 Human Molecular Genetics, 1998, Vol. 7, No. 11
a
b
Figure 1. Human ectodysplasin-A (EDA-A1) cDNA and protein. (a) Nucleotide sequence of EDA-A1. The 3′-untranslated sequence is not shown (GenBank accession
no. AF040628). Boundaries of each individual exon are indicated by black arrows. A white arrow indicates the internal splice donor site used in EDA-A2 transcripts
(GenBank accession no. AF061189). The residues of the transmembrane domain are underlined. A potential N-glycosylation site is boxed and the cysteine residues
at the C-terminus are circled. The Gly-X-Y triplets are shadowed and the positively charged domain is in bold. (b) Amino acid comparison of the human EDA-A1
protein and its mouse Ta homologue. Alignment was performed using the FASTA program (GCG package). Dots denote identical amino acids.
represent a new subgroup among the membrane-associated
collagenous type II transmembrane proteins. Previously known
members of this group include the MARCO protein (8), type XIII
and XVII collagens, the macrophage scavenger receptor and
complement component C1q (9).
We report here an extended analysis of the alternative human
forms of EDA gene transcripts, their expression pattern, the gene
structure and the first biochemical properties of the longest
protein isoform, designated ectodysplasin-A (EDA-A). We have
also identified novel mutations associated with the EDA
syndrome that argue for a functional role of the collagenous
domain of the EDA-A protein.
RESULTS
Characterization of EDA transcripts
We screened a human fetal brain cDNA library by hybridization
with a mouse cDNA probe corresponding to the longest Ta
isoform, Ta-A (6). We isolated nine cDNA clones and sequence
analysis confirmed that they correspond to a new EDA transcript
with additional exons. Sequence comparison in the GenBank
database allowed identification of three additional clones that
extend the 3′-end sequence of our longest human cDNA clone.
We assembled a consensus sequence (5307 bp) (Fig. 1a) that
1663
Human
Genetics,
1998,
7, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 11 1663
Figure 2. The EDA gene and alternatively spliced transcipts. Exons are shown as boxes, numbered from the 5′-end of the gene and the length of each exon (bp) is
marked under the box. Shaded boxes indicate sequences coding for (Gly-X-Y) triplets. The initiation and termination codons and poly(A) signals are also indicated.
The relative order of exons 1c, 1d and 1e is not known.
contains a 1176 bp open reading frame which is 91.4% identical
to the coding region of the murine Ta-A transcript. A 3889 bp
3′-untranslated region follows the termination codon and
contains a polyadenylation site (AATAAA) and a poly(A) tail at
its 3′-end. Based on its great similarity to the mouse Ta-A
sequence, the human transcript was named EDA-A1. EDA-A1
mRNA is predicted to encode a 391 amino acid protein that is
94.6% identical to the mouse Ta-A protein (Fig. 1b). As in Ta-A,
the deduced EDA-A1 amino acid sequence contains a
collagenous domain consisting of 19 Gly-X-Y triplets with one
disruption in the repeat structure. The three cysteines at the
C-terminal domain of the Ta-A protein are also conserved in
EDA-A1 (residues 332, 346 and 352), as well as a putative site for
N-glycosylation. A positively charged domain was identified
between amino acids 152 and 178, with a high content of
arginines and lysines (Fig. 1a).
The variation of transcribed EDA sequences was further
investigated by PCR amplification of 16 human cDNA libraries
from eight different tissues, using forward primers from exon 1
and reverse primers derived from vector sequences. After two
rounds of nested PCR, several bands were observed in all
libraries, ranging from 600 bp to 4 kb. Cloning of the PCR
products and sequencing revealed that they represent eight classes
of EDA cDNA clones, including EDA-O (2), EDA-B (6) and
EDA-A1 (Fig. 2).
A cDNA identical to EDA-A1 but missing nt 1161–1166 was
amplified from several libraries. This alternatively spliced
transcript was termed EDA-A2 and is predicted to encode a 389
amino acid protein that lacks residues Val307 and Glu308, both
present in EDA-A1.
Similarly to Ta-C (6), the EDA-C variant arises by an alternative
splicing event in which a portion of intron 1 (exon 1b) is included
in the transcript. However, in humans there is an earlier stop codon
in the intron sequence, resulting in a protein of only 147 residues
instead of the 220 amino acid protein predicted for Ta-C.
EDA-D is essentially identical to the EDA-B variant (6), except
that an additional 51 bp sequence (exon 1d) is found between
exons 1 and 3. The insertion introduces an early in-frame stop
codon after residue 142. Similarly, in EDA-E, inclusion of an 84
bp sequence (exon 1e) derived from an L1 repetitive element
between exons 1 and 2 results in insertion of an in-frame
termination codon after residue 148.
Finally, a faint band of 1.3 kb was cloned from a lung cDNA
library. This novel cDNA variant, EDA-F, has a 36 bp in-frame
insertion (exon 1c) between exons 1 and 2 and incorporates 135
bp of intron 2 (exon 2b) into exon 2 as a result of usage of an
alternate acceptor site within intron 2. The EDA-F transcript
would encode a 147 amino acid protein.
All EDA mRNAs appear to originate from alternative splicing
events that preserve exon 1. Thus, their deduced protein sequences
contain a common N-terminus and transmembrane domain and are
likely to belong to the class II transmembrane proteins, as described
for EDA-O (2) and Ta-A (6,7). All these new EDA variants were
screened against all available protein databases. Besides the very
high similarity within the human and mouse ectodysplasin-A
family, no other significant homologies were found other than the
strong matches expected between the Gly-X-Y domains and
various collagens from humans and other species.
Genomic organization
The position of the EDA exons and the sequences of exon–intron
boundaries were determined from YACs 3583, 1839, 1340 and
1891 (10) by ligation-mediated PCR, using sense or antisense
primers derived from the EDA-A1 cDNA sequence. The entire
EDA gene consists of at least 12 separate exons (Fig. 2). PCR
experiments indicate that the last exon spans from nt 1166 to at
least nt 3512. As can be seen in Table 1, all the donor and acceptor
splice junctions are in good agreement with the consensus
sequences for splice sites (11). We found an additional internal
splice donor in exon 8 that would produce the EDA-A2 variant,
which lacks exon 8b. A very similar genomic organization was
deduced for the mouse Ta gene (A.K. Srivastava et al., manuscript
in preparation).
1664 Human Molecular Genetics, 1998, Vol. 7, No. 11
Figure 3. Location of the EDA exons on the X chromosome genetic map. YACs are shown as horizontal bars. Positions of markers, STSs and EDA exons are indicated
by thin vertical lines. The orientation of the X chromosome is indicated (Xcen–qter).
Table 1. Nucleotide sequences at exon–intron boundaries of the EDA gene (GenBank accession nos AF060777–AF060788)
Exon
Intron
Intron
cttgagggatggggcggggtcggccgggacctcctccttcATTCC
E1
ACCAGgtgagtcacctagtaggggcggcggcggccccctcccctc
tggggtcaacctttgactaatgtacttgtaatttttacagATGGC
E3
AGATGgtaagtctactcagttgatcctttatcacttctgaattat
atttccaatgacttaattatctattttatttttcttatagGCCCA
E4
AAAGGgtaagttcctgactttataaaattgctgtcttgtcatata
cgttttcaaaaaaaaagtaacactgaatcctatttttcagGAAAG
E5
TTCTGgtgagttcccctgtctctccaccccaccaggtgcctttaa
aaaagattatgccctctgattgtcctatcctattttgcagGTGCT
E6
ACCAGgttggctggggattgctctcttcctgggtaggagggaaag
tactcatagtgactactctctatccttctcatcctgccagCCAGC
E7
GAATGgtaagaatcaaaataggctctctcccaaagaggagcttct
tctgacatgtactgagtgactgcccttctctcatactgagATCTT
E8
TAGAAgtgagtacggtcttaggcctaactcttcttatatccaaaa
tcctctcttccccaatcccttcttgttgcctctcactcagGTATA
E9
Exon sequences are indicated by upper case letters and flanking intron sequences by lower case letters.
Table 2. Mutations identified in the EDA gene
Patient
Mutation
Nucleotide change
Exon
Predicted effect on protein
Maternal carrier status
EDA 4
Arg156Cys
C708T
3
Missense
Sample not available
EDA 6
Gly291Arg
G1113A
8
Missense
Sample not available
EDA 5
Gly291Trp
G1113T
8
Missense
Sample not available
EDA 7
Asp298His
G1134C
8
Missense
Sample not available
EDA 14
Gly299Ser
G1137A
8
Missense
Sample not available
EDA 13
302dup8
Insertion of GGGAGCGA at 302
1
Termination after 38 residues
Carrier
EDA 3
836/841insC
Insertion of C at 836–841
5
Termination after 38 residues
Non-carrier
EDA10
801/814del18
Deletion of 18 bp at 801–814
5
In-frame deletion (Gly-X-Y)2
Carrier
EDA 2
891/901del18
Deletion of 18 bp at 891–901
5
In-frame deletion (Gly-X-Y)2
Carrier
EDA 15
789/795del36
Deletion of 36 bp at 789–795
5
In-frame deletion (Gly-X-Y)4
Carrier
EDA 11
IVS5-2A→G
A→G at 948-2
IVS5
Altered splicing
Carrier
EDA 9
del exon 3
3
Termination after 111 residues
Sample not available
We developed STSs for each exon and determined the STS
content in overlapping YACs (Fig. 3). Exons 5–9 were found to
be clustered in ∼6 kb of genomic DNA. BLAST nucleotide
searches with the sequences corresponding to exons 1c, 1d and 1e
revealed that they are contained in a 220 kb BAC clone
(AC002416) from Xq13 which also encompasses exons 1a and
1b. Exon 2b is included in a 93 kb BAC clone (AF003528) and
is contiguous with exon 2a.
Mutations in EDA patients
Based on the intron–exon organization, we initiated a search for
mutations on the newly discovered EDA coding sequences. We
designed primers flanking all EDA-A1 exons. DNA from 15 male
EDA patients was amplified by PCR and screened for mutations
by SSCP. As a result, we identified 12 different mutations (Table
2): two causing a frameshift (302dup8 and 836/841insC), three
in-frame deletions (see below), one splice site alteration
(IVS5-2A→G), five missense mutations (Arg156Cys,
Gly291Arg, Gly291Trp, Asp298His and Gly299Ser) and one
large deletion including exon 3.
PCR amplification of exon 5 revealed fragments of the
expected size in control individuals, but fragments which were
∼10–40 bp shorter in three unrelated EDA patients. Direct
sequencing of the PCR products revealed that patients EDA 2 and
EDA 10 carry two different in-frame 18 bp deletions that remove
1665
Human
Genetics,
1998,
7, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 11 1665
Figure 4. Sequence data of three EDA-causing mutations that remove Gly-X-Y repeats without interrupting the reading frame. (a) Direct nucleotide sequence of exon
5 in EDA patients 10, 2 and 15. The sequences span the breakpoint of the 18 bp (patients 10 and 2) or 36 bp (patient 15) deletions. In patients 10 and 2 the sense sequence
is shown, while in the bottom panel (patient 15) the antisense strand is depicted. Direct repeats are indicated with boxes. The nucleotide position in the cDNA is indicated
with arrows. (b) Schematic diagram of unequal crossing over as the putative mechanism for generation of the mutant alleles. In each case two copies of wild-type exon
5 sequences are shown. Blue and yellow rectangles mark the direct repeats involved in the putative misalignments. X marks the region of exchange. The bottom line
shows the sequence of the deleted allele that would result from the recombinational event. Note that the regularity of the (Gly-X-Y) repeats is not altered in the mutant
alleles.
six amino acids but do not disrupt the regularity of the Gly-X-Y
repeats. Similarly, a 36 bp deletion removes four Gly-X-Y triplets
in patient 15 (Fig. 4a and b). The exact boundaries of the deletions
could not be determined precisely because several alternative
deletions would result in the observed sequence difference. In
patients 10 and 2 the deleted region is located between nt 801–814
and 818–831 and between nt 891–901 and 908–918, respectively.
In patient 15 the deletion spans from nt 789–795 to nt 824–830.
All three deletions are flanked by two direct repeats of 13 (patient
10), 10 (patient 2) or 6 bp (patient 15).
No PCR products were obtained when amplifying DNA from
patient 9 with several combinations of primers from exon 3 and
flanking introns. This fact strongly suggests the presence of a
gross DNA rearrangement in this patient.
None of the deletions, insertions or missense mutations
described here was found among 75 unrelated, populationmatched control individuals, indicating that they do not represent
common polymorphisms. A 2 bp deletion at position +8 of the 5′
splice site of exon 5 (IVS5+8delCC) was found in some EDA
patients as well as in healthy controls. The allele frequency of this
polymorphism is 0.46. Another non-pathogenic nucleotide
substitution (IVS4+27T→A) was found in two patients and one
control individual.
Expression patterns of alternatively spliced EDA forms
We used RT–PCR to investigate the tissue distribution of the
splicing variants. A forward primer from the end of exon 1 was
paired with each of the seven primers derived from the
type-specific segments of the different transcripts. Each of the
seven primer sets was used to amplify single-stranded cDNA
from eight different tissues and several human cell lines. The
nucleotide sequence of the PCR products was confirmed by DNA
sequencing.
The results are summarized in Figure 5a and an example of
RT–PCR analysis is shown in Figure 5b. All tissues analyzed
express EDA-A transcripts, although at low levels. Sequencing
revealed that in all samples both EDA-A1 and EDA-A2 were
amplified. Transcripts for the EDA-O, B and C variants were also
detected in most tissues. The highest expression levels were
found in kidney, heart, liver and pancreas, whereas the lowest
levels were observed in brain and placenta. These results are in
agreement with those from previous northern blotting
experiments (2). Interestingly, expression of the EDA-D variant
showed clear tissue-specific differences and was restricted to
heart, liver, placenta and skeletal muscle. Very low to
1666 Human Molecular Genetics, 1998, Vol. 7, No. 11
a
undetectable levels of EDA-E and F were observed in all tissues
analyzed.
The suggested variable transcription of the EDA-A isoform
was verified by northern analysis. A northern blot with mRNAs
from four fetal tissues was hybridized with a probe specific for the
EDA-A variant (Fig. 5c). An mRNA species of the expected size
(5.3 kb) was detected in all tissues. The strongest signal was
observed in skin and small intestine.
Distribution of EDA-A protein in transfected cells
Cultured MCF-7 and COS-1 cells were transiently transfected
either with a construct coding for EDA-A2 or with the pCMV5
vector only. After 2 days, cells were permeabilized with Triton
X-100 and stained with anti-EDA antibodies (3).
Immunofluorescence analysis by laser confocal microscopy
shows a similar staining pattern for EDA-A2 as was previously
observed for EDA-O (3). In some of the transfected cells, a strong
signal was detected at the cell membrane, whereas in other cells
EDA-A2 appears as complex fine networks, compatible with
localization in the endoplasmic reticulum (Fig. 6a). In addition,
EDA-A2 expression in MCF-7 cells is associated with
morphological changes similar to those observed for EDA-O.
Cells positive for EDA-A2 that show membrane staining are
rounded-up, in contrast to the untransfected, flattened cells with
cytoplasmic extensions (Fig. 6b). No signal was seen in cells
transfected with the pCMV5 vector alone.
b
DISCUSSION
The results of this report have many implications. First, it
substantiates the previous suggestion of alternative transcription
of the EDA gene (2) and includes the full sequence of the longest
protein form and several alternative isoforms; second, it allows
mutation searches in the vast majority of patients with clinical
consequences; third, it indicates functionally important domains
of the protein, including the short collagenous segment; finally,
it verifies and extends the finding of a cell shape change induced
by overexpressing EDA protein (3).
The first identified transcript (here called EDA-O) was
encoded by two exons separated by 200 kb of genomic DNA (2).
A third human exon was identified by its similarity to the recently
identified mouse Tabby gene (6). Here we report the identification
of eight alternative forms. Their detailed characterization defines
nine new exons, raising the total number of separate EDA gene
exons to 12; four of these consist of continuous segments in
genomic DNA that may undergo alternative splicing at internal
splice donor sites. Three new exons map between the original
exons 1 and 2 and here are called 1c, 1d and 1e, in order to avoid
confusion generated by the repeated revision of exon numbering.
Several bands were evident in previous northern blotting
experiments with an exon 1-containing probe and mRNA from
either human (2) or mouse tissues (5,6). We have now confirmed
that the variation that results from alternative splicing events
affects the extracellular domain of the predicted polypeptide,
leaving the N-terminal domain constant. All transcripts but
EDA-A1 and EDA-A2 encode for truncated proteins lacking the
second collagenous domain. They all consist of 132 amino acids
derived from the common exon 1, followed by 3–46 amino acids
specific for each variant. The two longest transcripts, EDA-A1
and A2, differ from each other by just two amino acids that are
c
Figure 5. Analysis of alternatively spliced EDA transcripts. (a) Summary of the
expression pattern of the alternatively spliced EDA transcripts in human tissues
(upper) and cell lines (lower). +/– denotes the presence or absence of PCR
product, a blank space means not assayed. (b) RT–PCR of total RNA from eight
adult tissues. PCR was performed with a forward primer from exon 1 paired
with a reverse primer from exon 9 (EDA-A), exon 2 (EDA-O) or exon 1d
(EDA-D). A control RT–PCR using G3PDH primers is shown in the lower
panel. The sizes of the PCR products are shown in brackets. M, φX174 marker
(Promega). (c) Northern blot analysis of the expression of EDA-A transcripts.
mRNA from different fetal tissues and adult lung was hybridized with a PCR
product containing exons 8 and 9 specific for EDA-A transcripts.
1667
Human
Genetics,
1998,
7, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 11 1667
Figure 6. Immunolocalization of EDA-A2 in transfected COS-1 (a) and
MCF-7 (b) cells by confocal laser scanning microscopy. Cells were transfected
with the pCMV5-EDA-A2 construct and stained with anti-EDA antibodies
(green) and phalloidin–TRITC (red) for filamentous actin. EDA protein
localizes to the cell membrane in some transfected cells (a and b). MCF-7 cells
with membrane-associated staining show a rounded, detached morphology (b).
either included or excluded, dependent on the alternative usage of
splice donor sites in exon 8. The longer EDA-A1 form
corresponds most closely to the Ta-A form, both being of equal
length. Because we do not know the function of the EDA protein,
we can only speculate about the significance of the complex
alternative splicing pattern. It could reflect a high proportion of
aberrant splicing events, maybe due to the unusual size of intron
1. Alternative splicing could also constitute a mechanism to
regulate the amount of mature functional EDA protein produced.
Several previous studies indicate that alternative splicing may be
a rather common strategy to control gene expression (12,13).
Finally, alternative exon usage may provide a mechanism to
generate functionally distinct forms of the protein, as described
for many extracellular matrix proteins (14). The fact that
alternative splicing of the same exons has been reported in the
mouse Tabby gene (6) suggests that it may be biologically
significant.
The results reported in this paper show clearly that the EDA-A
isoforms are functionally important and critical for EDA
pathology. First, there is a striking amino acid conservation
between EDA-A1 and Ta-A: being 94.6% identical overall,
which rises to almost 99% if one excludes the 132 residues
encoded by exon 1. In addition, we have identified nine different
EDA-causing mutations in exons 5, 6 and 8, which are
exclusively present in the EDA-A transcript. Three mutations
removed a small number of the Gly-X-Y repeats without
interrupting the reading frame. They could have arisen by unequal
crossing over at any of the nucleotides of the flanking GC-rich
repeated sequences or, alternatively, they might result from a
replication error. Interestingly, similar Gly-X-Y deletions have
been described in several collagen genes, where they cause a
variety of clinical phenotypes (reviewed in ref. 15). The Gly-X-Y
domains of these collagens are known to form large triple helices.
Most of these disorders are dominantly inherited and it is thought
that molecules with shortened Gly-X-Y domains are likely to
interfere with normal triple helix formation and thus would have
a dominant negative effect (16). At present, we do not know if the
EDA-A protein, with a maximum length of 11 Gly-X-Y
uninterrupted repeats, is capable of forming triple helical
molecules; however, this domain does seem to be critical for the
correct developmental role of the protein. Four missense
mutations (Gly291Arg, Gly291Trp, Asp298His and Gly299Ser)
were found in exon 8. Interestingly, they are clustered in a nine
amino acid region (two of them affecting the same residue), thus
suggesting another putative functionally important domain.
Altogether, we have identified the molecular defect underlying
EDA pathology in 12 out of 15 patients. The remaining EDA
samples most probably carry mutations that escaped detection in
SSCP analysis. In addition, our study focused exclusively on the
coding region and therefore we could not detect regulatory
mutations in the promoter or mutations in introns that would
create cryptic splice sites. Misdiagnosis or mutations in as yet
unidentified alternatively spliced exons are less likely
explanations.
As one approach to explore the physiological meaning of EDA
isoform diversity, we have begun to characterize the tissue- and
cell-specific expression of individual mRNA isoforms. Despite
its low abundance (38 PCR cycles were needed for its detection),
most EDA transcripts have a wide tissue distribution. Some
differences in the tissue expression pattern were found, especially
for the EDA-D transcript, but further experiments are needed to
study the spatial distribution of the protein. We have previously
studied the in situ expression of the EDA transcript in various
stages of human embryonic and fetal development and in benign
and malignant epithelial tumours, using an antisense probe that
detects parts of exons 1 and 2 (4,17). Immunohistochemistry with
an antibody raised against the entire 135 amino acid EDA-O
protein has also been performed successfully in fetal tissues, but
failed to detect the protein in adult tissues, possibly reflecting its
low levels (4). Since exon 1 and the 132 amino acids encoded by
it are common to all the different variants, it is likely that these
results have provided an overview of expression of all the
different forms. Future studies will have to target segments and
domains that are specific for the different forms in order to
produce a more detailed picture of EDA expression.
The EDA-O protein has been shown to be transported to the cell
membrane in two mammalian cell lines (3). In addition,
expression of the protein in the epithelial breast cancer cell line
MCF-7 is associated with rounding up of the cells. Our present
results extend this finding by showing that the transcript encoding
for 389 amino acids (EDA-A2) is translated to a protein that
integrates with the cell membrane and induces rounding of
transfected MCF-7 but not COS-1 cells. Further transfection
studies should be performed to assess if other truncated species
are processed to associate with the cell membrane.
The precise function of the EDA protein in cell biology is still
obscure, apart from the well-known clinical phenotype its
mutations cause. The transcript map of the gene and the new
mutations identified in this study will be crucial for further
investigations at the protein and cellular levels.
MATERIALS AND METHODS
Screening of cDNA libraries
A human fetal brain cDNA library (Stratagene) was screened by
hybridization with a mouse Ta-A cDNA probe (6). cDNA
libraries (Quick-screen human cDNA library panel; Clontech)
from eight different tissues (brain, heart, kidney, liver, lung,
pancreas, placenta and skeletal muscle) were screened by PCR
using different combinations of λ primers (Clontech) and
EDA-specific primers from exon 1 (E1ext, 5′-CCGGGCCTCAAGAGAGTGGATGT-3′; E1int, 5′-GAGGTGGAGCGCAGGGAACTCCT-3′). PCR was performed using the XL-PCR system
1668 Human Molecular Genetics, 1998, Vol. 7, No. 11
(Perkin-Elmer) in two rounds of amplification according to the
manufacturer’s instructions. After 30 cycles of amplification
(93C for 1 min and 65C for 10 min), PCR products were
column purified and 10% used as template in a 30 cycle PCR
(93C for 1 min and 60C for 10 min) using nested primers.
Sequencing and sequence analysis
Sequencing of cDNA clone inserts was performed manually using
the fmol DNA cycle sequencing system (Promega). PCR products
were purified either with QIAquick Gel Extraction or QIAquick
PCR Purification kits (Qiagen) and subsequently sequenced using
the Perkin-Elmer dye-terminator cycle sequencing kit. Reactions
were analyzed in an ABI 377 sequencer.
Sequence analysis was carried out using BLAST, FASTA and
BLOCKS programs from the GCG sequence analysis package (18).
Ligation-mediated PCR
Agarose beads containing yeast DNA from YAC clones 3583,
1839, 1340 and 1891 were digested with RsaI or AluI. Ligationmediated PCR was performed as described by Kere et al. (19)
with sense or antisense primers derived from the EDA-A1 cDNA
sequence and a linker primer.
Patients and mutation screening
DNA from previously diagnosed EDA patients was used. Genomic
DNA fragments of 150–250 bp corresponding to exons 1, 3 and
4–9 were amplified by PCR using the following oligonucleotide
primers: E1aF, 5′-CCGGGCCTCAAGAGAGTGGATG-3′;
E1aR, 5′-GGGGTGCCCGAGCCGCCAA-3′; E1bF, 5′-GGCGGGAACGTGGAGCCGAG-3′; E3F, 5′-TTATGTTGGCTATGACTGAGTGG-3′; E3R, 5′-CCACCATGCCCTACCAAGAAGGT-3′; E4F, 5′-TCTTGGGGATCCCTCCTAGT-3′; E4R,
5′-TACAAAAATCGCACTCTTGATTT-3′; E5F, 5′-AAAAAAGTAACACTGAATCCTATT-3′; E5R, 5′-CTGGTGGGGTGGAGAGACAG-3′; E6F, 5′-GTGCACTCTGACTCTTCCTCCA-3′;
E6R, 5′-CTACCCAGGAAGAGAGCAATCC-3′; E7F, 5′-CCATTACTCATAGTGACTACTCT-3′; E7R, 5′-GATTATTTGGAGGCTGGGGAG-3′; E8F, 5′-TGTTGCCTCGATTATTCTGACA-3′; E8R, 5′-CACCGGATTTGCATTTTGGATA-3′; E9F,
5′-TTCTGTCAATTCACCACAGGG-3′; E9R, 5′-TTAGAGGTTCTGGGAGTCCTGGC-3′. In brief, 100 ng DNA was amplified in a 50 µl PCR reaction containing 1× GeneAmp buffer
(Perkin-Elmer), 0.2 mM dNTPs, 25 pmol each primer and 3 U
AmpliTaq Gold (Perkin-Elmer). The cycling conditions were
94C for 10 min followed by 38 cycles of 94C for 1 min and
60C for 1 min. For SSCP analysis, 2 µl PCR product were run on
a 12% acrylamide gel at room temperature and visualized by silver
staining methods. PCR fragments which gave aberrant SSCP
patterns were subsequently purified and sequenced as described
above.
reverse transcribed into cDNA using random hexanucleotide
primers and SuperScript II (Life Technologies) and 10% was used
as template in a PCR reaction. A panel of normalized, first-strand
cDNA preparations derived from eight adult tissues (brain, heart,
kidney, liver, lung, pancreas, placenta and skeletal muscle) was
purchased from Clontech (MTC Panel I). For amplification of
cDNA, primer E1int from exon 1 was combined with primers
derived from the type-specific regions of the different mRNAs
(EDA-O, 5′-GACGCCAGGTGAAACAGCTCCCAC-3′; EDAA, 5′-ACACTGGGGCATTTCTCTGCG-3′; EDA-B, 5′-CAGAAGTGATAAAGGATCAA-3′; EDA-C, 5′-TCGCCAACTTTTCTCGAGGCAG-3′; EDA-D, 5′-GCTGACAGGTCACAGGCTTAGCA-3′; EDA-E, 5′-TTAGAATATCAGTGGATTTCTCAGCA-3′; EDA-F, 5′-TTCTCAGTGAAAAAGAAACATGGAG-3′). PCR assays were performed as described for mutation
screening experiments. Semi-quantitative amplification conditions
were established using the same templates and G3PDH control
primers (Clontech). The reproducibility of the experiments was
evaluated by replicative RNA extractions, cDNA synthesis and
PCR reactions.
A northern blot with equal amounts of mRNA loaded in each
lane was purchased from Invitrogen. The probe was a 496 bp PCR
product amplified from the EDA-A cDNA clone, with a forward
primer from exon 8 and a reverse primer from the 3′-untranslated
region. Hybridization was performed using the manufacturer’s
protocol (Invitrogen). The filter was exposed on X-Omat film
(Kodak) for 1 day.
Construction of an EDA-A2 expression vector,
transient transfection and confocal imaging
A PCR fragment of 4 kb corresponding to EDA-A2 was digested
with ApaI and BamHI. The pCMV5-EDA-O construct (3) was
cleaned of the 524 bp fragment corresponding to EDA-O by
digestion with the same enzymes. Both DNA fragments were
then ligated and recombinant clones were sequenced to verify that
the engineered pCMV5-EDA-A2 construct was correct.
MCF-7 and COS-1 cells were transiently transfected using
Lipofectin (Life Technologies) with either pCMV5-EDA-A2,
pCMV5-EDA-O or pCMV5 alone as a control, following the
manufacturer’s instructions. Forty-eight hours after transfection,
EDA-A expression was detected by immunofluorescence using
anti-D-EDA antibodies (0.3 µg/ml) and FITC-conjugated goat
anti-rabbit IgG (Sigma), as described by Ezer et al. (3). Filamentous actin was stained with 0.77 µM phalloidin–TRITC (Sigma).
Samples were imaged with an LSM 410 laserscan microscope
(Zeiss) using excitation wavelengths of 488 and 543 nm.
ACKNOWLEDGEMENTS
RT–PCR and northern hybridization
The Ultraspec-II RNA isolation system (Biotecx) was used to
extract total RNA from cell lines derived from human breast
carcinoma (MCF-7), fetal kidney (293), epidermoid carcinoma
(A431), skin keratinocytes (HaCat) and placenta (ATCC n CRL
7325) and from locally established lymphoblast, fibroblast and
neural crest derived cell lines. Aliquots of 1–2 µg total RNA were
We thank Riitta Lehtinen for excellent help in mutation analysis
and Marja Mikkola for helpful discussions. We thank patients for
their cooperation in donating blood samples for study. This study
was supported by the Sigrid Juselius Foundation, Academy of
Finland, to J.K. and institutional funds of the JCSRI to A.K.S.
M.B. is a recipient of an EU TMR fellowship.
1669
Human
Genetics,
1998,
7, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 11 1669
REFERENCES
1. McKusick, V.A. (1994) Mendelian Inheritance in Man. Catalogs of Human
Genes and Genetic Disorders, 11th edn. Johns Hopkins University Press,
Baltimore, MD.
2. Kere, J., Srivastava, A.K., Montonen, O., Zonana, J., Thomas, N., Ferguson,
B., Munoz, F., Morgan, D., Clarke, A., Baybayan, P., Ellson, Y.C., Ezer, S.,
Saarialho-Kere, U., de la Chapelle, A. and Schlessinger, D. (1996) X-linked
anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a
novel transmembrane protein. Nature Genet., 13, 409–416.
3. Ezer, S., Schlessinger, D., Srivastava, A. and Kere, J. (1997) Anhidrotic
Ectodermal Dysplasia (EDA) protein expressed in MCF-7 cells associates
with cell membrane and induces rounding. Hum. Mol. Genet., 6, 1581–1587.
4. Montonen, O., Ezer, S., Saarialho-Kere, U., Herva, R., Karjalainen-Lindsberg, M.L., Kaitila, I., Schlessinger, D., Srivastava, A., Thesleff, I. and Kere,
J. (1998) The gene defective in anhidrotic ectodermal dysplasia (EDA) is
expressed in the developing epithelium, neuroectoderm, thymus and bone. J.
Histochem. Cytochem., 46, 281–290.
5. Ferguson, B.M., Thomas, N.S.T., Munoz, F., Morgan, D., Clarke, A. and
Zonana, J. (1998) Scarcity of mutations detected in families with X-linked
hypohidrotic ectodermal dysplasia: diagnostic implications. J. Med. Genet.,
35, 112–115.
6. Srivastava, A.K., Pispa, J., Hartung, A.J., Du, Y., Ezer, S., Jenks, T., Shimada,
T., Pekkanen, M., Mikkola, M.L., Ko, M.S.H., Thesleff, I., Kere, J. and
Schlessinger, D. (1997) The Tabby phenotype is caused by mutation in a
mouse homologue of the EDA gene that reveals novel mouse and human
exons and encodes a protein (ectodysplasin-A) with collagenous domains.
Proc. Natl Acad. Sci. USA, 94, 13069–13074.
7. Ferguson, B.M., Brockdorff, N., Formstone, E., Ngyuen, T., Kronmiller, J.E.
and Zonana, J. (1997) Cloning of Tabby, the murine homolog of the human
EDA gene, evidence for a membrane-associated protein with a short
collagenous domain. Hum. Mol. Genet., 6, 1589–1594.
8. Elomaa, O., Kangas, M., Sahlberg, C., Tuukkanen, J., Sormunen, R., Liakka,
A., Thesleff, I., Kraal, G. and Tryggvason, K. (1995) Cloning of a novel
bacteria-binding receptor structurally related to scavenger receptors and
expressed in a subset of macrophages. Cell, 80, 603–609.
9. Pihlajaniemi, T. and Rehn, M. (1995) Two new collagen subgroups:
membrane-associated collagens and types XV and XVIII. Prog. Nucleic Acid
Res. Mol. Biol., 50, 225–262.
10. Srivastava, A., Montonen, O., Saarialho-Kere, U., Chen, E., Baybayan, P.,
Pispa, J., Limon, J., Schlessinger, D. and Kere, J. (1996) Fine mapping of the
EDA gene: a translocation breakpoint is associated with a CpG island that is
transcribed. Am. J. Hum. Genet., 58, 126–132.
11. Shapiro, M.B. and Senapathy, P. (1987) RNA splice junctions of different
classes of eukaryotes: sequence statistics and functional implications in gene
expression. Nucleic Acids Res., 15, 7155–7174.
12. Bingham, P.M., Chou, T.B., Mims, I. and Zachar, Z. (1988) On/off regulation
of gene expression at the level of splicing. Trends Genet., 4, 134–138.
13. Adams, M.D., Rudner, D.Z and Rio, D.C. (1996) Biochemistry and
regulation of pre-mRNA splicing. Curr. Opin. Cell Biol., 8, 331–339.
14. Boyd, C.D., Pierce, R.A., Schwarzbauer, J.E., Doege, K. and Sandell, L.J.
(1993) Alternate exon usage is a commonly used mechanism for increasing
coding diversity within genes coding for extracellular matrix proteins.
Matrix, 13, 457–469.
15. Kuivaniemi, H., Tromp, G. and Prockop, D.J. (1997) Mutations in fibrillar
collagens (types I, II, III and XI), fibril-associated collagen (type IX) and
network-forming collagen (type X) cause a spectrum of diseases of bone,
cartilage and blood vessels. Hum. Mutat., 9, 300–315.
16. Willing, M.C., Cohn, D.H., Starman, B., Holbrook, K.A., Greenberg, C.R.
and Byers, P.H. (1988) Heterozygosity for a large deletion in the alpha 2(I)
collagen gene has a dramatic effect on type I collagen secretion and produces
perinatal lethal osteogenesis imperfecta. J. Biol. Chem., 263, 8398–8404.
17. Montonen, O., Ezer, S., Laurikkala, J., Karjalainen-Lindsberg, M.L.,
Thesleff, I., Kere, J. and Saarialho-Kere, U. (1998) Expression of the
anhidrotic ectodermal dysplasia gene in skin is diminished in cancer and
correlates with E-cadherin production. Exp. Dermatol., in press.
18. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of
sequence analysis programs for the VAX. Nucleic Acid Res., 12, 387–395.
19. Kere, J., Nagaraja, R., Mumm, S., Ciccodicola, A., D’Urso, M. and
Schlessinger, D. (1992) Mapping human chromosomes by walking with
sequence-tagged sites from end fragments of yeast artificial chromosome
inserts. Genomics, 14, 241–248.