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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A PLEC isoform identified in skin, muscle, and heart
Katarzyna B. Gostyńska,1 Henny Lemmink,2* Jeroen Bremer,1* Hendri H. Pas,1 Miranda
Nijenhuis,1 Peter C. van den Akker,2 Richard J. Sinke,2 Marcel F. Jonkman,1 Anna M. G.
Pasmooij1
University of Groningen, University Medical Center Groningen, Center for Blistering Diseases, Departments of
Dermatology, 1 and Genetics, 2 Groningen, the Netherlands
*contributed equally to this work
Published as letter to the editor in Journal of Investigative Dermatology DOI: 10.1016/j. jid.2016.09.032
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Background
Mutations in the PLEC gene, cause basal epidermolysis bullosa simplex (EBS) in 8% of cases (Bolling et al., 2014) . PLEC encodes the ubiquitously present cytolinker protein plectin, which plays an important role in the hemidesmosome by connecting keratin filaments to the underlying integrin α6β4 subunit (Andra et al., 2003; Koster et al., 2003) . Plectin deficiency in skin results in intraepidermal skin cleavage in basal keratinocytes (McLean et al., 1996) . In humans, eight distinct plectin isoforms have been identified arising from tissue specific translation. The gene sequences encoding these eight isoforms 1, 1a, 1b, 1c, 1d, 1e, 1f and 1g differ in their first exons and the corresponding upstream regulatory sequences (Fuchs et al., 1999; Rezniczek et al., 2003) . Additionally, alternative splicing of exon 31 results in a rodless plectin variant, which has been identified in human keratinocytes and skeletal muscle plectin (Winter and Wiche, 2013; Ketema et al., 2015) .
Depending on the mutation in PLEC, EBS can occur with isolated skin disease as seen in EBS-Ogna (Koss-Harnes et al., 2002) or EBS-plectin 1a (Gostynska et al., 2015) , or associated with pyloric atresia (EBS-PA) (Natsuga et al., 2010) or muscular dystrophy (EBS-MD) (Smith et al., 1996) . Here, we present a novel alternative PLEC isoform expressed in skin, muscle and heart, which is the result of a thus far unreported alternative splicing event in exon 8.
Results
The proband (IV-4, EB 210-01, Figure S1) presented at 17 years of age with complaints of acral skin blistering since the age of 2 years. She was the daughter of consanguineous Moroccan parents. Acral blistering, subtle nail pitting of the hands, focal plantar hyperkeratosis, onychokeratosis and onycholysis were observed (Figure S2). At the time of consultation she had left-sided ptosis, which worsened throughout the day. Chewing and swallowing took more time than normal. She complained of muscle weakness in her shoulders and arms, because of which she was not able to brush her hair or lift small objects such as a hairbrush. Walking for short distances quickly resulted in fatigue within 10 minutes. There was no visible muscular atrophy of extremities. Neurological consultation and electromyography revealed signs of myopathy. In her family, only her older brother had suffered from the same skin complaints. Both her father and brother (IV-I) had died from sudden cardiac death at ages 43 and 19, respectively (Figure S1). Following her brother’s death, the proband and her older sister (IV-3) underwent cardiologic screening, which had disclosed a cardiomyopathy.
Immunofluorescence (IF) staining of lesional skin showed intraepidermal cleavage with very thin lining of keratin in the blister floor. Staining of intact skin with the monoclonal antibody HD121 (kind gift from Dr. K. Owaribe), recognizing the rod domain of plectin, revealed reduced staining along the basement membrane zone (BMZ) (Figure S3 a,b). Transmission electron microscopy (TEM) further visualized pseudojunctional
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cleavage with the plasma membrane and the lamina densa in the blister floor (Figure S3 c,d). Taken together, the data suggested a plectinopathy and PLEC as the candidate gene. Sanger sequence analysis of the PLEC gene (NM_000445.3) encompassing all exons, however, revealed no mutation. In parallel, the patient was screened for mutations in
KRT5, KRT14, DST, COL17A1, ITGA6, ITGB4, LAMA3, LAMB3, and LAMC2, and, because of the
familial cardiomyopathy, a set of 55 cardiomyopathy-related genes using our targeted next generation sequencing panel (See supplementary) (Sikkema-Raddatz et al., 2013) . No mutations were identified. Therefore, additional intronic sequences falling outside the standard exon-intron sequencing regions in PLEC were analyzed for mutations, which revealed a novel homozygous deletion in intron 8, c.906+19_40del. Five different splice site prediction programs predicted that neither does this deletion affect the intron 8 consensus splice site, nor does it introduce a novel splice site.
As PLEC was the major candidate gene, we subsequently performed RT-PCR analysis on RNA isolated from a patient’s skin biopsy using primers surrounding intron 8 (Figure 1). This revealed intron 8 retention leading to a frameshift and a premature termination codon (PTC), p.Val303_Pro313ins11*. Consequently, the homozygous intronic deletion was predicted to result in a complete loss of plectin. Additionally, a novel transcript was identified that was 12 nucleotides shorter than wildtype. This product was the result of the use of an alternative splice site 12 nucleotides upstream from the consensus exon 8 splice donor site. The resulting plectin protein was predicted to be four amino acids shorter in length, missing amino acids 299-302 when compared to wildtype plectin. Although these amino acids 299-302 are conserved, they are just outside the actin-binding domain of plectin (Litjens et al., 2003) (Figure 2d). This shorter mRNA transcript was also found in skin of healthy control individuals at the same level, and a quantification of the PCR showed no difference in mRNA transcript quantity (Figure 1a). Western blot analysis with HD121 antibody on patient keratinocytes and fibroblasts revealed 30% and 10% plectin protein expression, when compared to healthy control keratinocytes and fibroblasts, respectively (Figure 2b,c). RT-PCR analysis on RNA isolated from healthy human striated FIGURE 1 (Left): Alternative splicing in exon 8 of PLEC. (a) RT-PCR analysis of patient RNA isolated from
cultu-red patient keratinocytes showed two products on a 3% agarose gel (lane 2). The larger product of 223 kb con-tained the retention of shortened intron 8 of 59 bp. This intron retention results in a PTC, and absence of protein (p.Val303_Pro313ins11*). The smaller product of 152 kb had a deletion of 12 nucleotides, and results in a protein that is four amino acids shorter (p.Val299_Asn302del4). The smaller, alternatively spliced product was observed in all healthy controls as depicted in lanes 3-7. Pictured to the right is the quantification of the RT-PCR of the diffe-rent products. There was no difference in quantity of PLEC transcripts that arose from the 5’ alternative splice site between healthy controls and patient. The slight difference in ratio between PLEC transcripts that arose from the 5’ consensus splice site could be explained by nonsense mediated mRNA decay of the consensus spliced tran-script in the proband. (b) Schematic representation of the exon-intron junction of exons 8 and 9 of PLEC while depicting the described splice sites and intronic deletion seen in the proband. The intronic deletion is depicted in green lower case letters, whereas the consensus splice site is marked with a blue line, and the alternative splice site with a red line. The dotted blue lines indicate the position of unused splice sites in the transcript that has the intron retention. (c) The numbers in blue and red correspond with the RNA transcripts that are observed in the agarose gel in (a). (d) The numbers in blue and red correspond to (a).
muscle and myocardium samples showed also the presence of the alternatively spliced transcript (Figure 2a).
Figure 2: Alternative splicing is present in myocardium and striated muscle (a) RT-PCR analysis on agarose
gel (3%) on RNA isolated from cryopreserved unrelated healthy human skin, healthy human myocardium and healthy human striated muscle samples. Healthy controls used were unrelated to the proband. The alternatively spliced product is present in skin, cardiac and striated muscle samples of healthy controls. The heteroduplex product is a combination of wildtype product and alternatively spliced product.
(b) & (c) Western blot using monoclonal antibody HD121 of cell lysates from cultured keratinocytes (b) and
fibroblasts (c) derived from normal healthy control and the proband (IV-4). Arrows indicate full-length plectin (molecular mass ~500 kDa). Quantification of plectin expression by patients keratinocytes showed 30% expression, compared to healthy control keratinocytes (b), and 10% expression by patient fibroblasts, compared to healthy control fibroblasts (c). (d) Conservation of plectin residues 299-302, which are omitted in the novel
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Discussion
Here, we describe physiological alternative splicing of exon 8 resulting in a novel four amino acids shorter, yet in-frame, plectin isoform that is present in skin, myocardium and striated muscle of healthy human controls. Complete loss of full-length plectin and rodless plectin results in a lethal EBS-PA phenotype, whereas expression of only rodless plectin results in an EBS-MD phenotype (Pfendner and Uitto, 2005; Rezniczek et al., 2010; Winter and Wiche, 2013) . We believe our patient would have developed the more severe, lethal form, EBS-PA, if not for the presence of the alternative splice site allowing the formation of the shorter transcript, resulting in four amino acids shorter full-length plectin and rodless plectin. The shorter plectin in the patient does not however prevent the formation of progressively severe MD, as her muscle symptoms are gradually worsening. This could be due to the lower amount of plectin production compared to normal control skin, i.e. 30% in keratinocytes and 10% in fibroblasts. Alternatively, the shorter plectin could have a decreased functionality. Nevertheless, the shorter plectin is considered to be at least partially functional, as the patient did not have a lethal phenotype.
In conclusion, our results reveal the presence of a novel PLEC isoform due to alternative splicing of exon 8. The functionality of the produced shorter length plectin protein is suggested by a moderate EBS-MD phenotype in the presented patient.
Supplementary
FIGURE S2: CLINICAL CHARACTERISTICS OF EB 210-01. Focal plantar hyperkeratosis and haemorrhagic
blister and erosions (a-c). Onychodystrophy, onycholysis with discoloration seen affecting all toenails (d). On the dorsal side of hands erosions with crusts and hyperpigmentation are seen (e).
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Supplementary methods
Patient
The patient (EB 210-01) was referred to our clinic for further analysis and subtype classification of EB. All experiments performed were done with material obtained for diagnostic purposes, which did not require additional approval from the institutional ethical committee. The patient gave informed consent for publication of photographs and the use of tissue samples. She was seen with her mother who consented for molecular carrier analysis. All non-patient tissue samples were contributed and analyzed anonymously. All experiments were conducted according to the principles of the Declaration of Helsinki.
Immunofluorescence antigen mapping
Four-mm skin biopsies of fresh blisters and healthy skin under the left arm were taken for immunofluorescence antigen mapping of the proband, and processed as described before (17). Mouse monoclonal antibody HD121 (gift of Dr. K. Owaribe) was directed against the plectin rod domain.
Electron microscopy
Two-mm punch biopsies of perilesional and non-lesional skin were taken from the upper arm and fixed with 2% glutaraldehyde in a phosphate buffer. Afterwards, they were stained with 2% osmium tetroxide, prior to being embedded in epon, sectioned and stained with uranyl acetate. Ultra-thin sections were then examined as described previously (Jonkman et al., 1992) .
Molecular analysis
Genomic DNA was extracted from peripheral blood lymphocytes using standard laboratory methods. The PLEC gene including all 8 isoforms (1, 1a, 1b, 1c, 1d, 1e, 1f and 1g) was screened for mutations by sequencing analysis of all exons including exon-intron boundaries. For in silico analysis of the mutation Alamut®Visual software (version 2.6.1, alamut.interactive-biosoftware.com) with protein and splice site prediction programs was used.
FIGURE S3 (Bottom left): IMMUNOFLUORESCENCE STAINING AND TRANSMISSION ELECTRON MICROSCOPY. Immunofluorescence staining of non-lesional skin of EB 210-01. (a,b) Monoclonal antibody
HD121 against the rod domain of plectin with decreased staining along the BMZ when compared to normal human skin (NHS) in controls. (c,d) Transmission electron microscopy of skin samples from lesional skin of EB 210-01. Blister cavity with a pseudojunctional split with the lamina densa in the blister floor (c). Magnification of the red square in (c) (d). The blue asterisk indicates rest segments of the plasma membrane with rests of hemidesmosomes. The yellow asterisk displays broken off keratin filaments with incomplete insertion into the hemidesmosome.
Western blot
Western blots were performed as described previously (10). Lysates from cultured keratinocytes and fibroblasts were used from the proband and healthy controls as substrates. To calculate the relative protein contents of the extracts, serial dilutions were run by SDS-PAGE. After Blue Silver staining the intensities of the lanes, indicating the total amount of protein present, were calculated using Quantiscan® software. Serial dilutions of all extracts were then immunoblotted and plectin was visualized with antibody HD121. The relative plectin expression was then calculated from the intensity of the plectin band using Quantiscan® software.
DNA and RNA analysis
Genomic DNA was extracted from peripheral blood lymphocytes using standard laboratory methods. The PLEC gene including all 8 isoforms (1, 1a, 1b, 1c, 1d, 1e, 1f and 1g) were screened for mutations by sequencing analysis of all exons including exon-intron boundaries. For in silico analysis of the mutation Alamut®Visual software (version 2.6.1, alamut.interactive-biosoftware.com) with protein and splice site prediction programs was used. Total mRNA was isolated from keratinocytes, fibroblasts, and cryopreserved tissue, using the RNeasy Micro kit (Qiagen). A 20 cycli RT-PCR with primers surrounding exon 8 (Forward: 5’-CTACGTCTCGTCGCTGTATG -3’ Reverse: 5’-GGAACCTGCGTTCCTCAAAG -3’) was performed using 1.0 µg cDNA template, and separated on a 3% agarose gel. The raw image was captured using a Gel Doc XR+ system (Bio-Rad), prior to quantification using ImageJ.
Skin panel
List of 55 genes screened using the targeted next generation sequencing panel for inherited cardiomyopathies in the UMCG in 2014:
ABCC9, ACTC1, ACTN2, ANKDR1, BAG3, CALR3, CAV3, CRYAB, CSRP3, DES, DMD, DSC2, DSG2, DSP, DTNA, EMD, EYA4, GATAD1, GLA, JPH2, JUP, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYPN, MYOZ1, MYOZ2, NEXN, PKP2, PLN, PRKAG2, PSEN1, PSEN2, RBM20, RYR2, SCN5A, SGCD, SOD2, TAZ, TBX20, TCAP, TMEM43, TNNC1, TNNI3, TNNT2, TPM1, TTN, TXNRD2, VCL
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