Download Type XVII collagen gene mutations in junctional epidermolysis

Experimental dermatology • Review article
Type XVII collagen gene mutations in junctional epidermolysis
bullosa and prospects for gene therapy
J. W. Bauer and C. Lanschuetzer
Department of Dermatology, General Hospital Salzburg, Austria
Summary
Non-Herlitz junctional epidermolysis bullosa (nH-JEB) is caused predominantly by
mutations leading to premature stop codons on both alleles of the type XVII collagen
gene (COL17A1). The analysis of mutations in this gene has provided a means of
correlating genotype with phenotype of nH-JEB patients. The phenotype of nH-JEB is
characterized by generalized blistering of skin and mucous membranes with atrophic
scarring and nail dystrophy. Atrophic alopecia is a distinct feature of nH-JEB patients,
but one that is not associated with the severity of the disease at other sites. Enamel
hypoplasia and pitting of the teeth are also characteristic for nH-JEB and can be used to
facilitate the correct diagnosis in children with a blistering skin disease. Analysis of the
biological consequences of mutations in the COL17A1 gene has shown that most
patients lack type XVII collagen mRNA due to nonsense-mediated mRNA decay.
Patients with these mutations can therefore be a target for corrective gene therapy using
vectors coding for full-length type XVII collagen. Proof of principle for this approach has
recently been demonstrated. The analysis of naturally occurring phenomena of gene
correction in the COL17A1 gene provides evidence for other mechanisms of gene
correction in genetic diseases. For example, exclusion of an exon carrying a mutation
can lead to a milder phenotype of nH-JEB than predicted by the original mutation. In
addition, we have gained data suggesting that COL17A1 exons harbouring pathogenic
mutations can also be repaired by trans-splicing, i.e. aligning corrected RNA sequences
to introns in the vicinity of faulty exons in the COL17A1 premtRNA.
Introduction
Epidermolysis bullosa (EB) is the term applied to a
heterogeneous group of inherited skin disorders in which
minor trauma leads to blistering of the skin and mucous
membranes.1 Depending on the level of tissue cleavage
EB has been divided into three main groups: EB simplex
with blister formation occurring by disruption of the
basal keratinocytes, junctional EB (JEB) with cleavage in
the lamina lucida and dystrophic EB with cleavage below
the lamina densa of the dermo-epidermal basement
Correspondence: Johann Bauer, Department of Dermatology, General
Hospital Salzburg, Muellner Hauptstr. 48, A-5020 Salzburg, Austria.
Tel.: +43 662 4482 3042. Fax: +43 662 4482 3044.
E-mail: [email protected]
Accepted for publication 4 September 2002
membrane zone. Most JEB patients belong to two main
groups: Herlitz JEB and generalized atrophic benign EB
(GABEB). Patients diagnosed with the former disease
usually die within the first 2 years of life, whereas the
latter diagnosis is associated with a better prognosis and
a tendency of further improvement during life. Initial
observations describing reduced expression of bullous
pemphigoid antigen 2 (also termed type XVII collagen)
in patients suffering from GABEB2,3 were followed by the
identification of mutations in the gene COL17A1 coding
for type XVII collagen in GABEB patients.4 Since then a
large number of mutations in the COL17A1 gene have
been published (Table 1). In addition, it has been shown
that the phenotype of GABEB can also result from
mutations in the genes coding for the polypeptide chains
of laminin 5.5 Recently, the term GABEB has been
merged into the group of non-Herlitz JEB (nH-JEB).6
2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60
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Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
Affected exons ⁄ introns
Reference
Mutations leading to stop codons
R1226X ⁄ 4150insG
R1226X ⁄ 1706delA
4003delTC ⁄ 4003delTC
3514ins25
4003delTC ⁄ Q1403X
4003delTC ⁄ G803X
2944del5 ⁄ 2944del5
2944del5 ⁄ Q1023X
1706delA ⁄ R1226X
2342delG ⁄ 2342delG
Q1016X ⁄ Q1016X
R1226X ⁄ R1226X
R145X
520delAG ⁄ 520delAG
2965delG ⁄ 2965delG
2666delTT
G258X ⁄ G258X
3674insT ⁄ 4426insC
2881delA ⁄ 2881delA
4003delTC ⁄ 4003delTC partially 4080insGG
R795X ⁄ R795X
exon 51 ⁄ exon 52
exon 51 ⁄ exon 18
exon 52 ⁄ exon 52
exon 48
exon 52 ⁄ exon 53
exon 52 ⁄ exon 34
exon 43 ⁄ exon 43
exon 43 ⁄ exon 45
exon 18 ⁄ exon 51
exon 30 ⁄ exon 30
exon 45 ⁄ exon 45
exon 51 ⁄ exon 51
exon 8
exon 7 ⁄ exon 7
exon 43 ⁄ exon 43
exon 37
exon11 ⁄ exon11
exon 54 ⁄ exon 50
exon 41 ⁄ exon 41
exon 52 ⁄ exon 52 ⁄ exon 52
exon 33 ⁄ exon 33
4
35
36
17
37
37
7
7
23
38
13
13
13
39
39
39
40
41
42
24
16
Acceptor splice site mutations
2441–2 A fi G ⁄ 2441–2 A fi G
2441–1 G fi T
3053–1 G fi C
intron 31 ⁄ intron 31
intron 31
intron 44
43
44
27
Donor splice site mutations
3871+1 G fi C
4261+1 G fi C ⁄ 4261+1 G fi C
intron 51
intron 52 ⁄ intron 52
27
28
Missense mutations
G627V
R1303Q ⁄ R1303Q
V1026M
G539E
G633D
S265C ⁄ S265C
exon
exon
exon
exon
exon
exon
16
13
41
37
14
45
In-frame skipping mutation
Ile18del389 (158del1172)
exon 2–exon 15
11
Digenic mutation
L855X ⁄ R1226X plus R635X. (LAMB3 gene)
exon 37 ⁄ exon 51
46
23
52 ⁄ exon 52
46
18
23
11 ⁄ exon 11
Table 1 Published mutations in the
COL17A1 gene.
Each line documents the first report of a distinct mutation in COL17A1. According to the
convention, mutations are reported as either the change in amino acid (R1226X, for
example, indicates that the codon for arginine at number 1226 is replaced by a nonsense
codon) or a change in nucleotide sequence (4150insG, for example, indicates an insertion of
a guanine at position 4150 in the complementary DNA). In one family, mutations in the
LAMB3 and the COL17A1 gene have been found (digenic mutation). The mutations have
been sorted by the year of appearance in the literature.
Analysis of genotype)phenotype correlation
The COL17A1 gene comprises 56 distinct exons, which
span approximately 52 kb of the genome on the long
arm of chromosome 10. It encodes a polypeptide, the
a1(XVII) chain, consisting of an intracellular globular
domain, a transmembranous segment, and an extracel-
54
lular domain that contains 15 separate collagenous
subdomains, the largest consisting of 242 amino acids.7
The mutations reported in the COL17A1 gene have
allowed the establishment of a mutation database,
which facilitates the analysis of the effects of specific
mutations on the clinical presentation of nH-JEB
(Fig. 1). Stop codon mutations or mutations leading to
2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60
Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
Figure 1 Schematic representation of the type XVII collagen polypeptide, and positions of the mutations disclosed in the corresponding gene,
COL17A1, in families with nH-JEB. The type XVII collagen polypeptide, as deduced from the corresponding cDNA sequence, consists of
intracellular, transmembranous and extracellular domains. The amino-terminus is localized in the keratinocyte, the extracellular
domain traverses the lamina lucida of the cutaneous basement membrane zone and the carboxy-terminus is situated in the lamina densa of
the cutaneous basement membrane zone. The arrows indicate the approximate position of mutations along the type XVII collagen
polypeptide chain. Mutations leading to stop codons are depicted above the polypeptide; all other mutations are shown below. The figure is
not drawn to scale.
downstream stop codons on both alleles are associated
with the original ‘GABEB’ phenotype.8 This phenotype is
characterized by blisters occurring since birth, which
heal with atrophic hyperpigmented or hypopigmented
scars. There is no severe scarring or exuberant granulation tissue. Nail dystrophy is seen in most of the
patients to a varying degree and some patients also
present with EB naevi.9 Mucosal involvement is variable
but less severe than in dystrophic EB (for a comprehensive review of ‘GABEB’ see reference 10). In one family, a
phenotype comparable to EB simplex has been described.
The underlying mutation in that case deleted a major
part of the intracellular domain of type XVII collagen.11
A distinct clinical sign of nH-JEB is atrophic, incomplete universal alopecia. However, one patient has been
described who has the characteristic clinical, ultrastructural and biochemical features of nH-JEB without
alopecia.12 nH-JEB in another patient with normal hair
and a mild phenotype with acral blistering has been
associated with the homozygous missense mutation
R1303Q.13 Patients with compound heterozygosity for
the stop codon mutation R145X and the missense
mutation G633D have been shown to present as a mild
form of nH-JEB including normal scalp hair, but missing
axillary and pubic hair.14 A remarkable variety in the
expression of alopecia has been noted in the reports of
Mazzanti et al.15 and Ruzzi et al.16 describing Italian
families supported by appropriate clinical, ultrastructural and molecular data. In these families homozygosity for the nonsense mutation R795X leads to a mild
phenotype of nH-JEB by exclusion of the faulty exon 33.
There is a spectrum of severity of the alopecia ranging
from almost complete universal alopecia to just absent
pubic and sparse axillary hair, suggesting that additional factors influence hair loss in these families.
Therefore, in general, alopecia cannot be used as a
marker of disease severity in nH-JEB.
Another feature, which is unique to nH-JEB, is
enamel hypoplasia and pitting of the teeth associated
with severe caries. These dental abnormalities are
2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60
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Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
present in patients with stop codon mutations or
mutations leading to downstream stop codons on both
alleles of COL17A1 or LAMB3 (coding for the b chain of
laminin 5) and can also be caused by hemizygosity for
the dominant negative missense mutation G627V.17
The teeth phenotype can be used as a guide to the
correct diagnosis in children with a blistering skin
disease.18
Gene therapy
Gene therapy describes the possibility of correcting the
consequences of a genetic defect on the molecular level.
Different methods are currently being tested for application in EB gene therapy (Table 2). Most commonly,
the correct expression of a defective gene in the skin has
been achieved by using retroviral delivery systems.
These retroviruses infect keratinocytes and stably
integrate DNA into the host genome. For the COL17A1
gene, phenotypic reversion of the ultrastructural characteristics of nH-JEB keratinocytes has been demonstrated using this method.19
Nevertheless, in the field of gene therapy there remain
multiple issues that have not yet been convincingly
resolved and prevent immediate translation to patients.
First, there is need to target keratinocyte stem cells to
achieve sustained expression of the transgene. This issue
is complicated by difficulties in identifying keratinocyte
stem cells by distinctive markers, although recently
better methods have been elaborated that identify stemcell-like fractions in mouse keratinocyte populations.20
Table 2 Current approaches in EB gene therapy.
Method
of transduction
Method of
correction
Retrovirus
cDNA**
PAC*** Clon
Transient
Transient
Transposase
Integrase
Gene
Trans-splicing
Ribozyme
cDNA
cDNA
Gene*
Reference
COL17A1
LAMB3
ITGB4
COL7A1
COL17A1
KRT14
LAMB3
COL7A1
19
47, 48
49
50
34
A
B
C
*Genes: COL17A1, coding for type XVII collagen; LAMB3, coding
for the b-chain of laminin 5; ITGB4, coding for b4 integrin;
COL7A1, coding for type VII collagen; KRT14, coding for
keratin 14.
**cDNA, complementary DNA.
***PAC, P1-based artificial chromosome.
A
McLean I and Terron A. J Invest Dermatol 2002; 119: 268.
B
Ortiz S et al. J Invest Dermatol 2002;119: 230.
C
Ortiz S et al. J Invest Dermatol 2002; 119: 343.
56
In addition, it has been shown that through ex vivo
selection using clonal analysis transduced keratinocytes
can be obtained that have the capacity to replicate for
more than 30 generations and are able to reconstitute a
fully differentiated epidermis on nude mice for more
than 40 weeks.21 Second, the ex vivo approaches to
gene therapy necessitate the transfer of transduced
epidermal sheets back to the patient’s skin using
surgical procedures with perhaps the potential for
scarring, infection or delayed wound healing. Possibly
these hurdles can be overcome through technical
advances such as programmed diathermosurgery
(Timedsurgery)22 or laser surgery. Finally, transduction
of keratinocytes with vectors containing normal fulllength cDNA copies of a defective gene is not indicated
in autosomal dominant forms of EB. In these forms of
EB, technologies such as ribozyme therapy or antisense
oligonucleotides are more relevant and might be used to
reduce the amounts of defective protein.
Analysis of naturally occurring phenomena of gene
correction in the COL17A1 gene pave the way for
alternative approaches to medical gene correction. In
1997 Jonkman et al.23 reported a patient with patches
of normal appearing skin in a symmetrical leaf-like
pattern on the upper extremities. The underlying
mutations in the COL17A1 gene were identified as
R1226X paternally and 1706delA maternally. In the
clinically unaffected areas of skin, about 50% of the
basal cells were expressing type XVII collagen protein,
albeit at a reduced level. The reason for the
re-expression of type XVII collagen was a mitotic gene
conversion surrounding the maternal mutation, thus
overriding the heterozygous mutation. These observations suggest that less than 50% of full-length type XVII
collagen is necessary to correct the phenotypic expression of nH-JEB. However, in a second nH-JEB patient, in
whom the homozygous mutation 4003delTC was partially corrected by the somatic mosaic back mutation
4080insGG, no phenotypic correction, i.e. no clinically
normal skin, could be observed. It is likely that the 25
incorrect amino acids between the deletion and insertion mutations prevented functional correction in this
patient.24 In a third example, a partly successful
naturally occurring attempt at gene correction by
modulation of splicing reactions has been described in
patients with the homozygous mutation R785X.16 In
these patients, exclusion of exon 33 harbouring this
mutation leads to an unusual mild phenotype, although
there is only a 5% level of detectable type XVII collagen
protein. Similar in-frame skipping of exons has also been
reported for patients with mutations in the COL7A1 and
LAMB3 genes.25
2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60
Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
Future perspectives
In recent years data have accumulated that indicate
that the spliceosome can be a target for attempting gene
correction. The spliceosome of a cell is the site where
small-nuclear ribonucleoproteins facilitate removal of
introns from premtRNA (the so-called splicing reaction)
and therefore control the correct assembly of exons in
mature RNA. If mutations disrupt the normal sequence
of a splice site, the spliceosome may use other intronic
or exonic splice sites (so-called cryptic splice sites)
usually leading to out-of-frame transcripts, and rarely to
in-frame transcripts. In this case, antisense oligonucleotide technology can be used to suppress the use of
cryptic splice sites. This approach has already been
tested successfully for the suppression of aberrant
splicing in patients with thalassaemia.26 This technology could be applied in type XVII collagen-deficient EB
patients, who have a splice site mutation producing at
least one alternative in-frame transcript.27,28 In this
case suppression of splice sites leading to out-of-frame
transcripts could redirect the splicing machinery to the
in-frame splice reactions, therefore producing polypeptides that have only small changes in protein composition. Another mechanism of ‘corrective’ splicing is the
activation of naturally occurring splice variants to
exclude faulty exons (see above) or to include missing
exons. This approach has been described for inclusion of
exon 7 in the survival motor neurone 2 gene in vitro. By
this mechanism spinal muscular atrophy patients could
be protected from the effects of loss of function in the
survival motor neurone 1 gene.29
Figure 2 SMaRT can reprogramme exons at the 3¢, 5¢ or within the gene. The COL17A1 premtRNA (target) is shown by a rod. The
premtRNA is the intermediate product of the RNA polymerases that transcribe the DNA of the gene into premtRNA in the nucleus. Shortly
after transcription the premtRNA still contains the intronic sequences, which are cleaved by a process called splicing, i.e. before the mature
RNA enters the cytoplasm for translation. For clarity, the introns surrounding the targeted exon, exon 52, are depicted separately. The
position of a common Austrian mutation 4003delTC in exon 52 is indicated. The Pre-Therapeutic-Molecule (PTM) is introduced into the
keratinocyte harbouring the mutation in the COL17A1 gene through a given transduction process. This RNA usually consists of a binding
region complementary to an intronic sequence and the exon(s) to be corrected. In the upper panel the binding region is complementary to
intronic sequences in intron 51, preceding exon 52. The remaining part of the PTM in the upper panel consists of RNA sequences
containing exons 52–56. PTMs successfully binding to intron 51 in COL17A1 premtRNA are replacing exon 52 harbouring the 4003delTC
mutation and the remaining C-terminus of COL17A1 RNA. This reaction is called 3¢ trans-splicing. In the middle panel the PTM replaces
exon 52 to the N-terminal end (5¢ trans-splicing). Also exon 52 alone can be replaced by trans-splicing (lower panel).
2003 Blackwell Publishing Ltd • Clinical and Experimental Dermatology, 28, 53–60
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Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
Modulation of splicing activities can also be attempted by trans-splicing. Trans-splicing refers to a process
whereby an intron of one premtRNA interacts with an
intron of a second premtRNA, enhancing the recombination of splice sites between these two premtRNAs.
A practical application of targeted trans-splicing to
modify specific target RNAs has been used in group I
ribozyme-based mechanisms. For example, using the
Tetrahymena group I ribozyme, targeted trans-splicing
has been achieved in human erythrocyte precursors to
alter mutant b-globin transcripts from individuals with
sickle cell disease.30 A related approach is spliceosome-mediated RNA trans-splicing (SMaRT). SMaRT
uses the introns preceding or following an exon
harbouring a mutation as a hinge to introduce a
corrected species of RNA into a given premtRNA (see
Fig. 2). Therefore, this technique reduces the size of a
corrective insert into a viral vector depending on the
position of the mutation. This fact is of particular
interest in genes coding for large proteins involved in
EB, i.e. type VII collagen (9.2 kB mRNA) and plectin
(14.2 kB mRNA). Proof of principle for SMaRT has
first been demonstrated in the b-human chorionic
gonadotrophin gene 6.31 Its applicability in the
correction of genetic diseases has been expanded by
work showing correction of the predominant cystic
fibrosis transmembrane conductance regulator (CFTR)
mutation deltaF508 in a CFTR-minigene construct
in vitro32 and in a CFTR mouse model in vivo.33
Recently, it has been demonstrated that also keratinocytes allow for trans-splicing reactions in the
COL17A1 gene34 and are therefore a potential therapeutic target for SMaRT.
Conclusion
Cloning of the COL17A1 gene and detection of mutations in this gene have facilitated the establishment of
the correct diagnosis and prognostic factors for nH-JEB
patients. Identification of the mutations in these patients
is the basis for correcting the genetic defects in the
COL17A1 gene. Analysis of naturally occurring phenomena of gene correction in the COL17A1 gene has
suggested that gene therapy is feasible to correct the
effects of such mutations. It has already been shown
that the reversal of phenotypic features of type XVII
collagen-deficient nH-JEB keratinocytes is achievable by
retroviral transduction. Besides introducing full-length
COL17A1 cDNA into keratinocytes, modulation of
splicing reactions in keratinocytes might also play a
role in gene therapy for inherited dermatological
diseases.
58
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collagen type XVII (COL17A1) locus in generalised atrophic
benign epidermolysis bullosa. Hum Genet 1997; 100:
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39 Floeth M, Fiedorowicz J, Schacke H et al. Novel homozygous and compound heterozygous COL17A1 mutations
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40 Shimizu H, Takizawa Y, Pulkkinen L et al. The 97 kDa
linear IgA bullous dermatosis antigen is not expressed in a
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bullosa with a novel homozygous G258X mutation in
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41 Pulkkinen L, Uitto J. Hemidesmosomal variants of epidermolysis bullosa. Mutations in the a6b4 integrin and the
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Type XVII collagen gene • J. W. Bauer and C. Lanschuetzer
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Key points
• Most mutations in the COL17A1 gene lead to premature
stop codons associated with nonsense-mediated mRNA
decay.
• Missense mutations in the COL17A1 gene are associated
with a milder phenotype.
• Incomplete alopecia universalis is a distinct feature for
nH-JEB, but is not a marker of overall severity.
• Enamel hypoplasia and pitting of the teeth is a distinct
clinical feature of nH-JEB that can be used to distinguish
nH-JEB from other EB subtypes.
60
• Phenotypic reversion of the ultrastructural features of
nH-JEB has been reported by retroviral expression of type
XVII collagen.
• Modulation of RNA splicing represents a new option for
gene correction in keratinocytes.
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