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col7a1 ekzon 12-24 delesyon literatürü kern2009

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C L I N I C A L A N D LA B O R A T O R Y I N V E S T I G A T I O N S
BJD
British Journal of Dermatology
Forty-two novel COL7A1 mutations and the role of a
frequent single nucleotide polymorphism in the MMP1
promoter in modulation of disease severity in a large
European dystrophic epidermolysis bullosa cohort
J.S. Kern,* G. Grüninger,* R. Imsak,* M.L. Müller,* H. Schumann,* D. Kiritsi,* S. Emmert,§ W. Borozdin,–
J. Kohlhase,– L. Bruckner-Tuderman*,** and C. Has*
*Department of Dermatology, University Medical Center Freiburg, Hauptstr. 7, 79104 Freiburg, Germany
Faculty of Biology, Spemann Graduate School of Biology and Medicine and **Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg,
Germany
§Department of Dermatology, University of Göttingen, Göttingen, Germany
–Center for Human Genetics Freiburg, Freiburg, Germany
Summary
Correspondence
Cristina Has.
E-mail: [email protected]
Accepted for publication
26 May 2009
Key words
collagen VII, large deletion, skin blistering,
splice site mutation
Conflicts of interest
None declared.
DOI 10.1111/j.1365-2133.2009.09333.x
Background Dystrophic epidermolysis bullosa (DEB) is a severe genetic skin blistering disorder caused by mutations in the gene COL7A1, encoding collagen VII.
Recently, the MMP1 promoter single nucleotide polymorphism (SNP) rs1799750,
designated as 1G 2G, was shown to be involved in modulation of disease severity
in patients with recessive DEB (RDEB), and was proposed as a genetic modifier.
Objectives To identify the molecular basis of DEB in 103 individuals and to replicate the results of the MMP1 promoter SNP analysis in an independent patient
group, as verification is necessary in such a rare and heterogeneous disorder.
Methods To determine the molecular basis of the disease, we performed COL7A1
mutation screening, reverse transcription–polymerase chain reaction (PCR) and
real-time quantitative PCR. The status of the MMP1 SNP was analysed by PCR and
restriction enzyme digestion and verified by sequencing.
Results We disclosed 42 novel COL7A1 mutations, including the first large genomic
deletion of 4 kb affecting only the COL7A1 gene, and three apparently silent mutations affecting splicing. Even though the frequency of the high-risk allele was
increased in patients with RDEB, no statistically significant correlation between
disease severity and genotype could be made. Also, no correlation was observed
with development of squamous cell carcinoma, a severe complication of DEB.
Conclusions Taken together, the results suggest that the MMP1 SNP is not the sole
disease modifier in different forms of DEB, and other genetic and environmental
factors contribute to the clinical phenotype.
Dystrophic epidermolysis bullosa (DEB) is a heritable skin fragility disorder, characterized by trauma-induced blister formation and healing with scarring.1,2 All subtypes are caused by
mutations in the COL7A1 gene encoding collagen VII.3 The
functionality and ⁄or the amount of this extracellular matrix
(ECM) protein – the main component of the anchoring fibrils
– are altered in DEB skin. This leads to a relatively broad spectrum of clinical phenotypes.4,5 The clinical severity of DEB
depends in part on the nature and location of the COL7A1
mutations and on their consequences on mRNA and protein
level.3,6–10 Still, established genotype–phenotype correlations
do not hold true for all affected individuals, and there are also
significant interindividual differences in patients harbouring
identical COL7A1 mutations. Even though more than 430
COL7A1 mutations have been reported in the literature10
(http://www.hgmd.cf.ac.uk/), the rate of novel mutations
detected in patients is still high. Moreover, sometimes the
consequences of mutations are hard to recognize and expression studies or predictions must be performed. Phenotypic
variation has been attributed to genetic modifiers, environmental factors, random events, and interactions between any
of these sources in general.11 Recently, a polymorphism in the
matrix metalloproteinase 1 (MMP1) gene promoter was proposed as the first genetic disease modifier of DEB.12
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
1089
1090 Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al.
MMP1 is a major proteinase of the matrix metalloproteinase
(MMP) family that degrades several ECM components, specifically type I collagen and other fibrillar collagens, including
collagen VII.13 The involvement of MMP1 in the pathology of
DEB has been suspected for a long time.14,15 Increased activity
of MMP1, MMP2, MMP3 and MMP9 and decreased tissue
inhibitor of metalloproteinase (TIMP)-1 have been detected in
the skin of patients more severely affected with DEB. Therefore, it was predicted that phenotypic variability in DEB could
be related to interindividual differences in MMP, specifically
MMP1, activity.16 The expression level of MMPs in intact skin
is very low, but is enhanced under pathological and regenerative conditions, such as inflammation, tumours, wound healing and tissue remodelling. The activity is regulated at three
levels: (i) by transcriptional control of MMP genes; (ii) by
proteolytic activation; and (iii) by small biological tissue
inhibitors (TIMPs).17
The frequent functional single nucleotide polymorphism
(SNP) in the MMP1 promoter, )1607del ⁄insG (rs1799750,
designated as 1G 2G), modulates the transcription of the
MMP1 gene, as the 2G allele forms a recognition site for Ets
transcription factor.18 In a French cohort comprising 31
patients with DEB, two genotypes, homozygous 2G (2G ⁄2G)
and heterozygous (1G ⁄2G), were associated with a more
severe recessive DEB (RDEB) phenotype.12 Almaani et al.19 did
not find evidence for the involvement of this SNP in DEB
pruriginosa in a group of 27 patients. Instead, they found a
possible association of the 2G allele with early-onset squamous
cell carcinoma in six individuals with RDEB.
As this putative disease modifier could have an important
prognostic value and implications for the design of molecular
therapy approaches, we assessed its role in a large cohort of
European patients with DEB.
Materials and methods
Patients and clinical samples
One hundred and three patients with clinically and morphologically diagnosed DEB were investigated. All patients had caucasian heritage and originated mainly from Central Europe.
Following informed consent, ethylenediamine tetraacetic acid
(EDTA)-blood and skin samples were obtained. EDTA-blood
samples from 40 individuals originating from Central Europe
were obtained and used as a control group. The study was
approved by the ethics committee of the University of Freiburg.
COL7A1 mutation detection
Genomic DNA from index patients and parents was extracted
from EDTA-blood using the QiaAmp DNA mini kit (Qiagen,
Hilden, Germany). Amplification of all 118 COL7A1 exons
and exon ⁄intron boundaries and sequencing were performed
as previously described.9 Mutations were confirmed by
resequencing. For identification of the breakpoints of the
COL7A1 genomic deletion the following primers were used
for polymerase chain reaction (PCR) and sequencing: F
5¢-TACCTCCAGCGCTCTCCTTC-3¢ and R 5¢-TGGAGAAACACATCAGGGTG-3¢. The mutation c.5499C>T was verified in
100 control chromosomes by restriction endonuclease digestion with MwoI according to the manufacturer’s protocol. The
mutation c.6217)3C>A was verified in 150 control chromosomes by direct sequencing.
Cell culture and RNA analysis
Total RNA was extracted with QiaAmp RNA blood mini kit
(Qiagen) from subconfluent keratinocytes of patients 38, 44,
62, 92 and 100, and a normal control cultivated in Keratinocyte Growth Medium (Invitrogen, Karlsruhe, Germany).
Reverse transcription (RT) was performed with Advantage
RT-for-PCR Kit (BD Biosciences, Heidelberg, Germany) with
0Æ5 lg of total RNA by using oligo dT primers. For RT-PCR
of COL7A1 the following primers were used: F5: 5¢-CTGAGGAGCTGAAGCGAGTT-3¢ and R8: 5¢-GCTGTGGGCTGTGGTATTCT-3¢; F60: 5¢-CAGCAGGAGAAAAGGGTGAC-3¢ and R68:
5¢-CTGGGACACCAGGAAAACC-3¢, F89: 5¢-AGATGGTGCCAGTGGAAAAG-3¢ and R93: 5¢-GATCTCCCTTCACACCTGGA-3¢;
F10: 5¢-TCCTTTCCTGGAACTTGGTG-3¢ and R27: 5¢-CAGACCACTGACTGCCTGGT-3¢; F102: 5¢-GGATGGTGACAAAGGACCTC-3¢ and R110: 5¢-GCAGAGCCATCATTTCCACT, respectively.
PCR products were separated on 1Æ5% agarose gels, purified
from the gel with the Qiagen gel purification kit and
sequenced in both directions as described.9
Quantitative real-time polymerase chain reaction
Quantitative real-time PCR and SYBR-Green I detection method
was performed, as described.20,21 Primers for generation of
small amplicons corresponding to COL7A1 exons 10–11, 11–
12, 13–14, 18–19 and 23–24 were designed. Amplicons corresponding to exon 4 (hsall 4F ⁄R) of the SALL4 gene were used as
an internal reference. For real-time detection, the ABI Prism
7900 system and 384-well plates (both from PE Applied Biosystems, Norwalk, CT, U.S.A.) were used. Reactions contained
0Æ25 mmol L)1 of each primer and 5 lL of QuantiTect SYBR
Green PCR Master Mix (Qiagen) in a final volume of 10 lL.
Each run included samples of serially diluted control DNA
(40 ng, 20 ng, 10 ng, 5 ng) for the generation of standard
curves for each primer pair, and genomic DNA samples from
patients, parents and controls (25 ng). The PCR programme
consisted of 50 C for 2 min, 95 C for 15 min, and 40 cycles
of 94 C for 15 s, 54 C ⁄60 C for 15 s, and 72 C for 1 min.
To verify the specificity of the primers, melting curve analysis
of the amplicons was performed and the results were evaluated
as previously described.20
Analysis of the 1G 2G MMP1 gene polymorphism
A rapid method for genotype analysis of the MMP1 gene 1G
2G polymorphism was employed as described.12,22 To verify
the procedure, randomly selected samples were also sequenced
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al. 1091
using the primers: F 5¢-CCTCTGATGCCTCTGAGAAGA and R
5¢-TCCTCCCCTTATGGATTCCT for the PCR and sequencing
reaction. In all cases, the results obtained by restriction
enzyme digestion and sequencing were identical. Using SPSS
software (Chicago, IL, U.S.A.), allele frequencies were analysed with Fisher’s exact test.
Morphological analyses of the skin
In most cases, the morphological diagnosis of DEB was ascertained with immunofluorescence mapping of blistered DEB
skin using a panel of antibodies to components of the epidermal basement membrane zone or with transmission electron
microscopy.23 Indirect immunofluorescence staining was
performed as described and the sections were observed with
an Axiophot fluorescence microscope (Carl Zeiss, Jena,
Germany).9
Results
The spectrum of dystrophic epidermolysis bullosa and
disclosure of 42 novel COL7A1 mutations
Our cohort consisted of 103 patients: 33 with dominant DEB
(DDEB), 29 with RDEB-other (RDEB-O) and 41 with RDEBsevere generalized (RDEB-sev gen), as defined according to
the revised epidermolysis bullosa classification (Tables S1 and
S2; see Supporting information).1 Of the 103 patients, six had
the clinical presentation of DEB pruriginosa (patients 1, 6, 14,
23, 25 and 37); in five cases the inheritance was dominant
and in one recessive.24,25 Ten individuals with RDEB had one
or several squamous cell carcinomas (patients 62, 63, 82, 87,
88, 89, 95, 96, 101, 102; Table S2; see Supporting information). As expected, mutations in patients with DDEB and
RDEB-O resulted in the presence of low levels of collagen VII
in the skin (Tables S1 and S2; see Supporting information).
Mutation screening disclosed disease-causing mutations in
161 of the 163 expected mutated alleles. Forty-two novel
mutations were identified, comprising 10 glycine substitutions, three missense mutations other than glycine substitutions, seven nonsense, 11 deletion ⁄insertion, and 11 splice
site mutations (Table 1). The full mutation constellations of
all individuals are described in Tables S1 and S2 (see Supporting information). These data demonstrate the high variability
of mutations in DEB and contribute to the expanding COL7A1
mutation database.
The first intragenic large deletion in COL7A1
In the case of patient 92, using genomic DNA for amplification, no PCR products were obtained for exons 13–24. These
findings suggested the presence of a genomic deletion in a
homozygous state in the region. This was confirmed by quantitative real-time PCR, which demonstrated in the patient a
homozygous deletion, while in both parents the amount of
genomic DNA in the investigated region was consistent with
the status of heterozygous carrier of the deletion. To the best
of our knowledge, this is the first intragenic large genomic
deletion in COL7A1. The breakpoints have been identified and
demonstrated that it spans over 4 kb starting in intron 12
and ending in exon 24; the deletion was designated as
c.1637)240_3255del4064. A large deletion comprising 255–
520 kb encompassing nine to 15 genes including COL7A1 has
been described before in a patient with moderately severe
RDEB in compound heterozygous state with a splicing
mutation.26
Novel splice site mutations
The consequences of the novel splice site mutations were verified by mRNA studies and by predictions with the software
NetGene2 (http://www.cbs.dtu.dk/services/NetPGene/) or
FSPLICE (http://linux1.softberry.com) as shown in Table 2.
Interestingly, three apparently silent mutations emerged as
splice site mutations. In patient 44, sequencing of the entire
coding sequence and exon ⁄intron boundaries of COL7A1
revealed no sequence changes, except one novel sequence
variant, a homozygous transversion c.5499C>T in exon 64.
This converts the codon GGC for glycine into a synonymous
codon GGT at position 1833 (p.G1833G) (Fig. 1a). The
splice site prediction performed with the software NetGene2
provided no evidence for modification of splicing. The software FSPLICE predicted that the mutation introduced a novel
cryptic donor splice site in exon 64. RT-PCR with primers
located in exons 60 (F) and 68 (R) disclosed in the patient’s
sample a slightly lower band than the 426-bp band obtained
in the control (Fig. 1b). One additional minor band was
obtained in both the patient and the control. Sequencing
demonstrated that the patient’s major band resulted from
aberrant splicing of exon 64, which led to the deletion of
35 bp from the mRNA (designated on RNA level as
r.5497_5532del). A novel cryptic donor splice site was activated
at the site of the substitution )35 positions from the normal
donor splice site (Fig. 1c). This is predicted to cause a frame
shift starting with codon 1834, and formation of a premature
termination codon, 25 codons downstream (p.E1834RfsX25).
Both parents were heterozygous carriers of the mutation. The
sequence variant was confirmed in the patient’s DNA sample
and excluded from 100 control chromosomes by digestion with
restriction enzyme MwoI (not shown).
Two other apparent silent mutations, c.7017C>T,
p.G2339G and c.7023G>A, p.K2341K, were predicted to
interfere with the donor splice site of exon 90 (Fig. 1d,e).
They did not completely abolish the normal splice site, but
either created a novel donor splice site upstream (Fig. 1d), or
activated a cryptic donor splice site downstream (Fig. 1e),
respectively. In patient 62, the prediction regarding the mutation c.7023G>A was confirmed on the RNA level by RT-PCR.
Presumably, in vivo, both normal and aberrant transcripts
occur, resulting in RDEB-O in both patients.
Finally, the consequences of the mutation c.7929+1G>A,
which affects the conserved position +1 at the donor splice
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
1092 Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al.
No.
Location
exon ⁄ intron
COL7A1 mutation cDNA
Missense and nonsense mutations
1
22
2966G>A
2
32
3940G>A
3
60
5287C>T
4
66
5570G>A
5
73
6080C>T
6
73
6173G>C
7
74
6191G>T
8
75
6236G>T
9
78
6394G>A
10
84
6689G>A
11
85
6745C>T
12
88
6927G>C
13
96
7345G>A
14 101
7598G>A
15 102
7621C>T
16 103
7705G>A
17 108
7987G>T
18 111
8233C>T
19 112
8329C>T
20 117
8764C>T
Splice site mutations
21
6
846G>A
22
6
846+1G>A
23
18
2441)2A>G
24
19
2587+1G>A
25
64
5499C>T
26
74
6217)3C>A
27
85
6751)2_1delAG
28
90
7017C>T
29
90
7023G>A
30
91
7068+5G>A
31 106
7929+1G>A
Deletion ⁄ insertion mutations
32
13–24
1637)240_3255del4064
33
17
2305_2314delGTGAGGACTGinsTT
34
20
2638dupC
35
21
2789_2790dupAC
2791_2796del6
36
23
3020delC
37
27
3566delT
38
45
4590delA
39
78
6422delG
40
95
7289delC
41 105
7861_7865delCCCCG
42 111
8253_54dupAG
Table 1 Novel COL7A1 mutations identified in
this study
Collagen VII
protein ⁄
consequence
Phenotype
W989X
G1314R
R1763X
G1857E
P2027L
G2058A
G2064V
G2079V
G2132S
G2230E
Q2249X
K2309N
G2449R
G2533D
R2541X
G2569S
E2663X
R2745X
R2777X
R2922C
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
DDEB
DDEB
RDEB
DDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
a
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
a
a
a
a
a
a
a
a
a
a
PTC
V769FfsX2
E880GfsX23
R931TfsX3
RDEB
RDEB
RDEB
RDEB
P1007QfsX9
M1189SfsX76
G1531EfsX177
G2141EfsX64
P2430QfsX35
P2621GfsX4
V2752EfsX34
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB
RDEB, recessive dystrophic epidermolysis bullosa; DDEB, dominant dystrophic epidermolysis bullosa; PTC, premature termination codon. aSee Table 2.
site of exon 106, were verified by RT-PCR. This demonstrated
the presence of two transcripts: one resulted from skipping of
exon 106 leading to an in-frame deletion of 18 codons, and
the second resulted from inclusion of intron 106 into the
reading frame leading to a frame shift and premature termination codon (PTC) (Fig. 1f).
The 1G 2G MMP1 single nucleotide polymorphism
in patients with dystropic epidermolysis bullosa
and association with disease severity
The 1G 2G MMP1 SNP genotype distribution was consistent
with the Hardy–Weinberg equilibrium. The distribution of the
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al. 1093
Table 2 Splice site mutations discovered in this study and their consequences, as predicted by the splice-site predictor
Splice-site scorea
Location
c.DNA position
Normal
Mutant
Theoretical predictions at mRNA protein level
Exon 6 ds)1
Intron 6 ds+1
Intron 18 as)2
Intron 19 ds+1
Exon 64 ds)34
Intron 74 as)3
Intron 85 as)2_1
Exon 90 ds)7
Exon 90 ds)1
846G>A
846+1G>A
2441)2A>G
2587+1G>A
5499C>T (p.G1833G)
6217)3C>A
6751)2delAG
7017C>T (p.G2339G)
7023G>A (p.K2341K)
0Æ88
0Æ88
5Æ40b
0Æ55
11c
0Æ98
1Æ00
1Æ00
1Æ00
LDS
LDS
LAS
LDS
11
0Æ49
LAS
0Æ95
0Æ82
7068+5G>A
1Æ00
LDS
7929+1G>A
0Æ95
LDS
Skip exon 6, FS, PTC
Skip exon 6, FS, PTC, verified by RT-PCR
Skip exon 18, IF
Skip exon 19, IF
Novel ds upstream, FS, PTC, verified by RT-PCR
Skip exon 75, IF
Skip exon 85, IF
Novel ds upstream
Activation of cryptic ds downstream, verified by
RT-PCR, FS, PTC
(i) Skip exon 91, IF
(ii) Cryptic ds downstream
(i) Skip exon 106, IF, verified by RT-PCR
(ii) Inclusion of intron 106 into the mRNA, FS,
verified by RT-PCR
Intron 91 ds+5
Intron 106 ds+1
ds, donor splice site; as, acceptor splice site; LDS, loss of donor splice site; LAS, loss of acceptor splice site; FS, frame shift; PTC, premature
termination codon; IF, in-frame; RT-PCR, reverse transcription–polymerase chain reaction. aIf not mentioned, predictions were made with
NetGene2. bPredicted with FSPLICE, threshold 4Æ175. cPredicted with FSPLICE, threshold 6Æ099.
(a)
(c)
(d)
(e)
(f)
(b)
Fig 1. Consequences of splice site mutations. (a) Direct sequencing
of polymerase chain reaction (PCR) products from genomic DNA
disclosed in patient 44 (upper panel) a novel homozygous
transversion in exon 64: c.5499C>T which changed the codon GGC
in the wild-type sequence to the synonymous codon GGT at position
1833, p.G1833G. This GT induced a new donor splice site (red circle
in the upper panel). (b) In the upper panel, reverse transcription
(RT)-PCR analysis with primers spanning exon 60–68 of COL7A1
demonstrated in patient 44 (P44) a transcript which was slightly
smaller than the 426-bp transcript obtained in the control (Co). In the
lower panel, RT-PCR with control primers for GAPDH is shown. One
additional minor product was obtained in the patient and the control.
(c) Sequencing demonstrated that the shorter major RT-PCR product
in the patient resulted from aberrant splicing of exon 64 and skipping
of the last 35 nucleotides of exon 64 (upper panel). The control
sequence is shown in the lower panel. (d) Schematic representation of
the predicted consequences of the mutation c.7017C>T resulting in a
novel cryptic donor splice site (cds 1) in patient 53. The mutation
(purple and bold) induced a new donor splice site (red circle)
(NatGene2 score 1Æ00), while the score of the normal splice site
downstream (green circle) decreased slightly from 1Æ00 to 0Æ95. Both
splice sites might be active. (e) Schematic representation of the
predicted consequences of the mutation c.7023G>A resulting in
formation of a novel cryptic donor splice site (cds 2) in patient 62.
The mutation (purple and bold) leads to the decrease of the score of
the normal splice site (green circle) from 1Æ00 to 0Æ82, while the
score of the cryptic splice site (red circle) located downstream
increases from 0Æ7 to 0Æ9. Again, both donor splice sites might be
active. (f) RT-PCR and sequencing (not shown) demonstrated the
presence of two transcripts in patient 100 (P100) instead of the
correct transcript obtained in the control sample (Co). The schematic
representation shows that the abnormal transcripts resulted either
from skipping of exon 106, or from inclusion of intron 106 in the
mRNA.
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
1094 Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al.
1G/1G
EB
DD
1G/2G
en
O
B-
E
RD
2G/2G
vg
se
B-
E
RD
ls
tro
n
Co
Fig 2. Distribution of 1G ⁄ 1G, 1G ⁄ 2G and 2G ⁄ 2G genotypes in
patients with dystrophic epidermolysis bullosa (DEB) and controls.
The graph represents the percentage of patients and controls with
1G ⁄ 1G, 1G ⁄ 2G and 2G ⁄ 2G genotypes. DDEB, dominant DEB;
RDEB-O, recessive DEB-other; RDEB-sev gen, recessive DEB-severe
generalized.
three genotypes 1G ⁄1G, 1G ⁄2G and 2G ⁄2G in patients and
controls is shown in Figure 2 and Table 3. The major allele in
our control group was 1G (52Æ5%), as reported in the NCBI
SNP database for another control caucasian population
(56Æ7%). The allelic frequency of 2G in DDEB (45Æ5%) was
similar to our controls and to the NCBI control population
mentioned above (47Æ5% and 43Æ3%). Notably, in the RDEBO and the RDEB-sev gen groups the major allele was 2G
(58Æ6% and 52Æ4%), yet this increase, compared with controls,
was not statistically significant (P = 0Æ23 and P = 0Æ64;
Table 3). Interestingly, in the RDEB-O group, with all patients
having residual collagen VII in the skin, the increased 2G frequency and therefore increased transcription of MMP1 could
indeed play a role in degradation of the protein, and the status
of this frequent SNP might play a role in the modulation of
pathological processes in the skin.
Next, we analysed possible associations of the 1G 2G MMP1
SNP with clinical phenotypes. We compared generalized and
localized DDEB, and found no statistically significant difference
in the 2G allele frequency. Therefore, in this cohort of patients
with DDEB, the 1G 2G MMP1 SNP is not likely to act as a sole
or strong disease modifier.
We also compared the 1G 2G MMP1 SNP genotype of
patients with RDEB-O and RDEB-sev gen harbouring mutations that allow residual expression of collagen VII. The RDEBsev gen cohort did not show a significant increase in 2G allele
frequency, as compared with RDEB-O patients. In the group
with clinical DEB pruriginosa, the 1G ⁄2G and 2G ⁄2G genotypes were found in five of six patients.
Of 10 patients with RDEB who developed aggressive squamous cell carcinoma, four were homozygous 1G ⁄1G, three
were heterozygous 1G ⁄2G and three were homozygous
2G ⁄2G. Thus, an increased association of the 2G allele was
not found in this group of patients, and observations in this
cohort differ from those reported by Almaani et al.19
MMP1 and disease severity in individual cases
We further addressed the question of whether the 1G 2G
MMP1 SNP could correlate with disease severity in individual
situations. Four patients with RDEB-sev gen harboured COL7A1
mutations that allowed expression of collagen VII, in contrast
to all others who had mutations leading to a PTC and
complete absence of collagen VII in the skin. Patient 63 was
compound heterozygous for a splice site (c.425A>G) and a
missense (p.R2063W) mutation and was homozygous for the
MMP1 SNP 2G allele. He had severe disease and died at the
age of 34 years of aggressive squamous cell carcinoma. Patient
75 had a homozygous glycine substitution mutation
p.G2449R and was heterozygous (1G ⁄2G) for the MMP1 SNP.
He had an unexpectedly severe phenotype with mitten deformities. Predictions did not indicate any splicing abnormality.
Immunofluorescence mapping revealed a reduced collagen VII
signal at the dermal-epidermal junction. Patient 94 was homozygous for the mutation p.G1857E and for the MMP1 SNP 2G
allele, and had severe blistering at birth, but had not yet
developed severe scarring and mitten deformities, because of
the young age (1 year). Immunofluorescence mapping indicated strong reduction of collagen VII at the dermal-epidermal
junction. Predictions provided no evidence that the mutation
Table 3 MMP1 single nucleotide polymorphism genotype and allele frequency
Genotype
DDEB
RDEB-O
RDEB-sev gen
Controls
CAUC1a
1G ⁄ 1G
1G ⁄ 2G
2G ⁄ 2G
Total patient number
1G allele frequency
2G allele frequency
8 (24Æ2%)
20 (60Æ6%)
5 (15Æ2%)
33
54Æ5%
45Æ5%
1 (3Æ4%)
22 (75Æ9%)
6 (20Æ7%)
29
41Æ4%
58Æ6%
10 (24Æ4%)
19 (46Æ3%)
12 (29Æ3%)
41
47Æ6%
52Æ4%
6 (15%)
30 (75%)
4 (10%)
40
52Æ5%
47Æ5%
9 (30%)
16 (53Æ3%)
5 (16Æ7%)
30
56Æ7%
43Æ3%
DDEB, dominant dystrophic epidermolysis bullosa; RDEB-O, recessive dystrophic epidermolysis bullosa-other; RDEB-sev gen, recessive
dystrophic epidermolysis bullosa-severe generalized. aThirty individuals of caucasian heritage (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.
cgi?rs=1799750).
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al. 1095
In contrast, patient 85 had a severe phenotype comprising
extensive chronic wounds, knee and elbow joint as well as
hand contractures and webbing at the age of 9 years. Mucosal
involvement led to severe oesophageal strictures, necessitating
dilatation and placement of a percutaneous endoscopic gastrostomy tube, as well as to anal stenoses. He was compound heterozygous for the COL7A1 mutations p.[R1632X]+[E2059G]
and homozygous for the 1G allele (Fig. 3a,b). In comparison,
patient 34, the 56-year-old uncle of patient 85, homozygous
for p.E2059G and homozygous 2G ⁄2G, was relatively mildly
affected with RDEB-O (Fig. 3a,c). Collagen VII was decreased
in the skin of both individuals (Fig. 3d–g). Therefore, in this
constellation, it is likely that other disease modifiers account
for the different phenotypes.
(a)
(c)
(b)
Discussion
(d)
(e)
(f)
(g)
Fig 3. Clinical presentation and collagen VII expression in a family in
which the clinical severity does not seem to correlate with the 1G 2G
MMP1 single nucleotide polymorphism genotype. (a) Family pedigree
showing patient 85 (nephew) and patient 34 (uncle) with recessive
dystrophic epidermolysis bullosa and their COL7A1 and MMP1
genotypes. (b) Clinical presentation of the 9-year-old patient 85. The
upper panel shows scarring and contractures of the fingers. The lower
panel reveals extensive trauma-induced skin fragility of the back.
(c) The 56-year-old uncle of patient 34 shows a much milder clinical
appearance. The upper panel demonstrates atrophic scarring and
mild finger contractures. In the lower panel, individual erythematous
lesions on the skin of the back can be seen. (d–g) Indirect
immunofluorescence staining of the skin with the collagen VII
antibody LH7.2 demonstrates strong staining in control samples
(d,f), and reduced, but positive signals in the skin of patients 85
(e) and 34 (g). The asterisk indicates the blister.
could affect splicing. In all three cases, despite the presence of
missense mutations, the increased transcription rate of
MMP1 caused by the 2G allele present might contribute to the
more severe phenotypes by degradation of residual anchoring
fibrils.
Identifying sources of disease variation is crucial to improving
biologically valid therapeutic concepts for DEB. It is attractive
to consider a genetic variant, which increases MMP1 expression, as a modulator of disease severity. However, given the
relatively small numbers of patients available and the likely
low magnitude of effect size, replication of results in independent populations is essential. Furthermore, mutation analysis
and careful investigation of the consequences of mutations are
essential in establishing genotype–phenotype correlations. Our
results indicate that large genomic deletions and apparently
silent mutations might account for a number of cases of DEB,
and might be overlooked in a routine diagnostic procedure.
Moreover, as COL7A1 mutation analysis is laborious, usually
only the DNA of the index patient is analysed completely, and
the mutations are then confirmed in related individuals.
Therefore, within the same family the presence of other mutations or sequence variants explaining variability of phenotypes
cannot be excluded. The results of the mutation analysis in
our large cohort of 103 patients with DEB, disclosing 42 novel
mutations, emphasizes the complexity of molecular mechanisms underlying DEB. Screening for COL7A1 mutations
remains essential for understanding the pathogenesis of this
devastating disease.
In our cohort, patients with RDEB-O had an increased frequency of the 2G allele, but due to the limited number of
cases no statistical significance was observed. In patients with
RDEB-sev gen, the 2G allele might account for an increased
severity in individual cases, as demonstrated by the examples
of patients 63, 75 and 94, but it did not correlate with
occurrence of squamous cell carcinoma. In the DDEB group
we found no statistical association between the MMP1 genotypes and disease phenotype. In contrast to previously
reported data, in our small group of patients with DEB pruriginosa, the genotypes 1G ⁄2G and 2G ⁄2G were predominant.19 These observations highlight the difficulty of
obtaining sufficient statistical power to disclose real disease
modifiers in rare diseases with relatively few affected individuals. Taken together, we cannot find clear-cut correlations
between MMP1 promotor polymorphism and DEB phenotype
2009 The Authors
Journal Compilation 2009 British Association of Dermatologists • British Journal of Dermatology 2009 161, pp1089–1097
1096 Dystrophic epidermolysis bullosa and MMP1, J.S. Kern et al.
in our cohort of 103 patients. This question should be further addressed in a joint effort with as many patients with
DEB as possible, to clarify the putative disease-modifying role
of the MMP1 SNP. Moreover, measurement of the actual
expression and activity of MMP1 in the skin of patients as
well as the susceptibility of different mutated collagen VII
variants to degradation would be necessary to confirm predicted effects.
In vivo in the skin, the biological context is very complex
and the individual contribution of a modifier gene, like MMP1
in this case, remains unknown. In cutaneous wounds in which
the basement membrane is disrupted, as is the case with DEB,
skin injury induces temporally and spatially restricted expression of MMP1 in migrating keratinocytes within hours.17 This
expression is shut down after re-epithelialization is completed.
MMP1 is also expressed in fibroblasts in granulation tissue,
where it is believed to participate in remodelling of the
ECM.17 The regulation of MMP1 activity depends not only on
the expression levels, but also on activation of the proenzyme
and on inhibition, mainly by TIMPs. These processes are finetuned to assure normal wound healing. In DEB, the basement
membrane is repeatedly disrupted, and the chronic wound situation modifies the environment, leading to disturbances of
the balance between proteases and inhibitors. Therefore, one
might expect that a primary increase of MMP1 transcription
would be compensated by other mechanisms to maintain tissue homeostasis, e.g. variation in the expression and activity
of MMP activators and inhibitors, and that these might also
be implicated in DEB severity. Future functional studies must
clarify the partial roles of the 1G 2G MMP1 SNP and other
modifying factors in the complex interplay of cell–matrix
interactions in healing DEB skin.
Other modifier genes are certainly involved in modulation
of phenotypic variations in different forms of epidermolysis
bullosa.27 For example, the splicing machinery is known to
be a genetic modifier in single-gene disorders. Patients with
splicing mutations can have variations in the level of
correctly spliced RNA transcribed, which correlates with the
disease severity.28 In the case of missense mutations, mutant
proteins can be misfolded and subjected to intracellular
degradation, processes which are likely to be the subject of
interindividual genetic variations.29 New approaches such as
genetic modifier screens in model organisms or biodata
mining will help identify novel candidates for disease
modifiers in the future.30,31
Acknowledgments
We thank all patients who participated in this study, the
physicians who sent us samples and Martin Festag for help with
RNA-analysis. The work was supported by a Network Epidermolysis Bullosa grant from the Federal Ministry for Education
and Research to L.B.-T. and by the Excellence Initiative of the
German Federal and State Governments (Spemann Graduate
School of Biology and Medicine to J.S.K., and Freiburg Institute
for Advanced Studies, School of Life Sciences to L.B.-T.).
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Supporting information
Additional Supporting information may be found in the online
version of this article:
Table S1 COL7A1 mutations and MMP1 genotypes in patients
with dominant dystrophic epidermolysis bullosa
Table S2 COL7A1 mutations and MMP1 genotypes in patients
with recessive dystrophic epidermolysis bullosa
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by
the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
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