Exon skipping therapy for dystrophic epidermolysis bullosa
Bremer, Jeroen
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Publication date: 2018
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Bremer, J. (2018). Exon skipping therapy for dystrophic epidermolysis bullosa. University of Groningen.
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Amelioration of junctional epidermolysis bullosa due to
exon skipping
C. Kowalewski1, Jeroen Bremer2, A. Gostynski2, K. Wertheim-Tysarowska3, K. Wozniak1,
J. Bal3, M.F. Jonkman2, A.M.G. Pasmooij2
1Department of Dermatology and Immunodermatology, Medical University of Warsaw, Poland 2University of Groningen, University Medical Center Groningen, Department of Dermatology, Center for
Blistering Diseases, Groningen, the Netherlands
3Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland
Abstract
Mutations in the COL17A1 gene lead to the genetic blistering disorder junctional epidermolysis bullosa generalized intermediate type (JEB-gen-intermed). Antisense oligonucleotide-mediated exon skipping is a strategy that aims to skip the mutation-containing exon and thereby produce a smaller but functional protein. COL17A1 is an interesting candidate, as 53 of the 55 exons (96%) can be skipped without disturbing the reading frame. Information on the functionality of the shortened protein product is important in order to obtain support for this therapeutic strategy. Here we report a patient with JEB-gen-intermed with amelioration of the phenotype due to exon 49 skipping by two distinct mechanisms – premature termination codon-induced exon skipping and revertant mosaicism – both of which induced skipping of the same exon. The patient was compound heterozygous for two inherited COL17A1 mutations, a frameshift mutation in exon 18 (c.1490_1491delinsT, p.Ala497Valfs*23) and a nonsense mutation in exon 49 (c.3487G>T, p.Glu1163Ter). Upon clinical examination, skin patches were found that were resistant to blister formation. In these patches, naturally corrected cells were present that harboured an additional splice-site mutation, c.3419–1G>T, resulting in skipping of the mutation-containing exon 49. This natural gene therapy phenomenon shows that type XVII collagen with residues 1140–1169 deleted is largely functional. In addition, in affected skin cells a low level of exon 49 skipping was observed. Our results support the notion that skipping of a mutated in-frame exon in COL17A1 ameliorates the phenotype.
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Background
Absence of type XVII collagen (C17) protein due to mutations in the COL17A1 gene results in the genetic blistering disorder junctional epidermolysis bullosa generalized intermediate type (JEB-gen-intermed).1 Patients with JEB-gen-intermed have fragile skin and mucous
membranes from birth. Blistering develops with little or no trauma. The transmembrane protein C17 plays a key role in adhesion in the dermoepidermal junction.
Currently there is no cure for this devastating disease, although several therapies are under investigation, among which is antisense oligonucleotide (AON)-mediated exon skipping.2, 3 This new therapeutic strategy is based on the use of AONs that bind
to complementary sequences of the pre-mRNA. The aim is to induce skipping of the mutation-harbouring exon by modulation of pre-mRNA splicing, and thereby produce a slightly smaller, although functional, protein. In Duchenne muscular dystrophy this therapy is very promising and at the forefront of research with ongoing clinical phase III studies.4 The COL17A1 gene is an interesting candidate, as it has many small exons that can
be skipped without disturbing the reading frame: 53 of the 55 coding exons (96%). It is important to have information on the functionality of the shortened C17 product, and on the amount that is needed to result in correction of the phenotype. In this case we show functionality of C17 after exon 49 skipping, which was caused by two mechanisms: (i) premature termination codon-induced exon skipping and (ii) correction of the inherited mutation by a spontaneous somatic mutation, also referred to as revertant mosaicism.
Case report
A 21-year-old woman with JEB-gen-intermed presented clinically with total alopecia, rudimentary nails, enamel dental problems, oral erosions and disseminated tense blisters on the skin healing with atrophic scars. On the lower arm several nonblistering skin patches were observed, both with slight hyperpigmentation and ‘different sensation’, as compared with surrounding areas (Figure. 1). Analysis of blood genomic DNA identified compound heterozygosity for a frameshift mutation in exon 18 (c.1490_1491delinsT, p.Ala497Valfs*23) and a nonsense mutation in exon 49 (c.3487G>T, p.Glu1163Ter) of the
COL17A1 gene. The patient provided informed consent for the study.
To test for skin functionality the ballpoint test was used.5 In this rub test the tip
of a retracted ballpoint pen is pushed over the skin for a distance of 2–4 mm with a quick movement, and subsequently the presence or absence of blister formation can be observed under a dermatoscope. Punch biopsies of 4 mm were obtained for immunofluorescence microscopy from affected skin and clinically healthy skin without blister formation after the ballpoint test. Stainings were performed with three monoclonal antibodies for C17: VK4 (endodomain 381–399, exon 7), Lu-226 (epitope 1080–1107, exons 47–48) and 233 (epitope 1118–1143, exons 48–49).6, 7 The biopsy taken from affected skin fitted with a
and VK4 (Figure. 2a). The epitopes of 233 and Lu-226 are close to each other, and it was therefore unexpected that the staining of these two antibodies was different.
In the biopsy taken from clinically healthy skin a mosaic pattern was observed. Along 95% of the basement membrane zone the staining resembled the staining of the biopsy taken from affected skin. Interestingly, approximately 5% of the biopsy showed a stronger staining more comparable with normal human control skin for VK4 and Lu-226. However, 233 was still negative. The epitope of 233 consists of the amino acids of exons 48–49 (1118–1143).7 As the Lu-226 and 233 epitopes showed a different pattern, it was
expected that a somatic mutation had occurred in exons 48–49 resulting in loss of the 233 epitope.
Discussion
The ameliorated phenotype in revertant skin in our patient is due to the presence of C17 that is slightly smaller (30 amino acids missing from 1140–1169), although functional, as demonstrated by the ballpoint test performed in revertant skin of the patient's forearm. Knowing the threshold for C17 is essential for therapy options. Kiritsi et al.9 showed that
about 12–14% of the physiological C17 levels are sufficient to have a major influence on the clinical phenotype leading to only mild cutaneous involvement. Moreover, Ruzzi et
al.10 showed that as little as 3–4% can result in a mild phenotype. That the amount of C17
is important can also been seen in our patient. Despite the fact that the slightly smaller
Figure 1. Image of the patient's arm. The biopsy location is marked with an arrow in the slightly hyperpigmented revertant patch on the forearm. In the circle above the biopsy location a negative (i.e. no blister) ballpoint test was performed. Two other suspected revertant areas are also marked with dotted lines. The area closer to the hand is more hyperpigmented, likely due to more sun exposure.
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Serine/arginine-rich proteins bind to ESEs and thereby promote exon splicing. When using Human Splicing Finder Version 2·4·1 (http://www.umd.be/HSF/), which is a bioinformatics tool to predict splicing signals, the programme predicts two RESCUE-ESEs in exon 49: GGATCA (nucleotides 3425–3430) and GAGGAG (nucleotides 3484–3489, Figure. 3). When introducing the nonsense mutation c.3487G>T the second RESCUE-ESE is lost, which could account for the skipping of exon 49. The disturbance of an ESE site crucial for exon definition was also proposed by Covaciu et al.12 for exon 87 skipping in the COL7A1 gene
by the silent exonic c.6846G>C, p.Leu2282Leu mutation, which was localized 15 bp from the acceptor splice site.
Recent studies have indicated that the majority of patients with COL17A1
Figure 2. (a) Immunofluorescent staining of control and patient skin biopsies. Type XVII collagen protein (C17) is schematically displayed at the top, with the striped transmembrane domain. In red, amino acid 497 is indicated. In black are the in-frame skipped amino acids. Immunofluorescent staining of control skin, unaffected revertant skin and affected mutant skin with Lu-226, VK4 and 233 primary antibodies is shown. In the patient, mutant skin staining for C17 was reduced with Lu-226 and VK4, while 233 was absent. More specifically, Lu-226 stained 1 + for the basement membrane zone [BMZ; control skin: 4 + BMZ, 3 + intercellular staining (ICS)] and VK4 showed 1 + BMZ (control skin: 3 + BMZ, 2 + cytoplasmatic (CYT)]. In the revertant areas (indicated with white arrows) the staining was comparable with normal human control skin for Lu-226 (2 + BMZ, 1 + ICS) and VK4 (2/3 + BMZ, 2 + CYT), but still negative using 233, as the epitope of this antibody lies in exons 48–49.
(b) DNA sequence analysis of laser dissection microscopy samples surrounding exon 49. (A) Splice acceptor site of mutant keratinocytes; (B) mutant keratinocytes with the germline c.3487G>T mutation; (C) splice acceptor site of revertant keratinocytes with the correcting c.3419–1G>T mutation, which is absent in mutant keratinocytes; (D) revertant keratinocytes with the germline c.3487G>T mutation. (c) cDNA sequence analysis of healthy control and mutant patient keratinocytes. (A) Gel electrophoresis of cultured control (1) and mutant keratinocytes (2, 3) cDNA; (B) polymerase chain reaction (PCR) product 1 of control keratinocytes; (C) PCR product 2 of mutant keratinocytes harbouring the germline c.3487G>T mutation; (D) PCR product 3 of mutant keratinocytes showing exon 49 skipping. The asterisk in (A) indicates a heteroduplex product of exon 49 skipped and wild-type product (sequence data not shown).
gene therapy, lessons can be learned regarding the function of C17, as besides exon 49 skipping, deletion of exon 30 also leads to normally functioning protein (Table 1). Furthermore, the c.2237delG, p.Gly746AlafsTer53 COL17A1 mutation in exon 30 was corrected by multiple mechanisms within one patient. Also, the deletion of both exons 30 and 31 together led to correction. Finally, skipping of exon 33 by a constitutive process occurring at a very low level in all keratinocytes led to an unusually mild JEB phenotype.11
However, not all exon deletions result in a milder phenotype, as was shown by the deletion of exon 32 in patients with a more classical severe JEB-gen-intermed phenotype.14
The amino acids encoded by exon 32 are predicted to have a more important function. Generation and characterization of recombinant C17 lacking amino acids encoded by specific exons would provide more insight into the functionality of internally truncated C17 variants. Assays that would be helpful are trypsin digestion and binding-affinity measurements to binding partners of C17, such as laminin-332 and α6 integrin.
In conclusion, this is the first reported patient in whom two different mechanisms are observed – premature termination codon-induced exon skipping and revertant mosaicism –both of which lead to skipping of the same exon. The amount of C17 produced is crucial.
Figure 3. Schematic display of pre-mRNA splicing of exon 49. Wild-type exon 48–50 pre-mRNA splicing results in the joining of exons 48, 49 and 50. The small blue boxes in exon 49 represent predicted exonic splicing enhancer sequences (ESEs). Due to the germline nonsense mutation c.3487G>T (red rectangle), one of the predicted ESEs is lost. Consequently, next to the transcript bearing a premature termination codon, an exon 49-deleted mRNA variant is produced that is lacking the nonsense mutation, which is translated into a shortened type XVII collagen protein (C17). In the revertant keratinocytes the additional somatic splice-site mutation (c.3419–1G>T) indicated by the green rectangle is present. This additional splice-site mutation results in only exon 49-deleted mRNA. The exon 49-deleted mRNAs are translated into C17 lacking amino acids 1140–1169.
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Table 1. Review of published and unpublished studies of exon skipping in COL17A1.
Exon Mutaion Clinical
Phenotype Cause of milder phenotype Reference
30 (36 bp) c.2251C>T; p.Gln751Ter
Mild form of JEB-gen-intermed
Exon skipping most likely induced by premature termination codon. An additional mutation (c.2262 + 13T>G) was found in the intron 30 border but was predicted to have no effect on splicing. Protein production estimated to be 15% of normal
15
c.2237delG; p.Gly746AlafsTer53
Revertant area with clinically healthy phenotype Correcting mutation c.2263 + 2T>C at DNA level 13 Correcting mutation c.2228-101_2263 + 70delins15 at DNA level 13 Correcting mutation c.2259_2263 + 9del at DNA level
13 Correcting mutation c.2238C>T at DNA level 13 30 + 31 (108 bp) c.2237delG; p.Gly746AlafsTer53
Revertant area with clinically healthy phenotype.
Correcting mutation c.2227 + 153_2336–318del resulting in deletion of 2165 bp at DNA level
13
32 (27 bp) c.2336–2A>G Classical JEB-gen-intermed phenotype
Not applicable 14
33 (36 bp) c.2383C>T; p.R795Ter
Mild form of JEB-gen-intermed
Constitutive exon skipping occurring at a very low level. Protein production estimated to be 3–4% of normal
10
49 (90 bp) c.3487G>T, p.Glu1163Ter
Revertant area with clinically healthy phenotype
Correcting mutation c.3419–1G>T at DNA level
This thesis
Mild form of JEB-gen-intermed
Exon skipping at a very low level possibly because of disruption of an ESE site due to the c.3487G>T mutation
This thesis
JEB-gen-intermed, junctional epidermolysis bullosa generalized intermediate type; ESE, exonic splicing enhan-cer sequence.
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