- Email: [email protected]
A PLEC Isoform Identified in Skin, Muscle, and Heart
LETTER TO THE EDITOR
A PLEC Isoform Identified in Skin, Muscle, and Heart Journal of Investigative Dermatology (2016) -, -e-; doi:10.1016/j.jid.2016.09.032
TO THE EDITOR 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 a6b4 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 tissuespecific 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 (Ketema et al., 2015; Winter and Wiche, 2013). 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 (Natsuga et al., 2010) or muscular dystrophy (Smith et al., 1996). Here, we describe an alternative PLEC isoform expressed in skin, muscle, and heart, which is the result of a thus far unreported alternative splicing event in exon 8. The proband (IV-4, EB 210-01, Supplementary Figure S1 online) presented at 17 years of age with complaints of acral skin blistering since the age of 2 years. The proband gave written and informed consent for publication of clinical photographs and description. No institutional approval
was required as all performed methods were a part of routine clinical diagnostics. She was the daughter of consanguineous Moroccan parents. Acral blistering, subtle nail pitting of the hands, focal plantar hyperkeratosis, onychokeratosis, and onycholysis were observed (Supplementary Figure S2 online). 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 (Supplementary Figure S1). After her brother’s death, the proband and her older sister (IV-3) underwent cardiologic screening, which had disclosed a cardiomyopathy. Immunofluorescence 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 (Supplementary Figure S3a and b online). Transmission electron microscopy further visualized pseudojunctional cleavage with the plasma membrane and the lamina densa in the blister floor (Supplementary Figure S3c and d). Taken together, the data suggested a
Abbreviation: EBS, epidermolysis bullosa simplex Accepted manuscript published online 18 October 2016; corrected proof published online XXX XXXX ª 2016 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
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 (Supplementary Table S1 online) (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.906þ19_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 reverse transcription-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, 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 wild type. 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 299e302 when compared with wildtype plectin. Although these amino acids 299e302 are conserved, they are just outside the actin-binding domain of plectin (Litjens et al., 2003) (Figure 2d). www.jidonline.org
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ska et al. KB Gostyn
Shortened Human PLEC Isoform Total mRNA template (arbitrary units)
a
2 1 1
2
35.000 30.000 25.000 20.000 15.000 10.000 5.000 0
Control Patient (n=5) Consensus splicing
Control Patient (n=5) Alternative splicing
b AGGATGGG GTGAGGGCCAAC gtgagtggggggcccggagggcagggggcttgggcctggacgccccgacgacccctgacagccgcccgtgcctgcccgcag GAGCTGCAGCT Intron 8 Exon 9 Exon 8
c
Healthy individual 1
standard splicing Intron 8 (81bp)
Exon 8 1
Exon 9
alternate splicing Intron 8 (81bp)
Exon 8
Exon 9
Patient IV-4 2
PTC
standard splicing Exon 8
2
alternate splicing Intron 8 (59bp)
Patient IV-4 (2)
Patient IV-4 (2)
Healthy individual (1) Healthy individual (1)
Exon 8
d
Exon 9
Intron 8 (59bp)
Exon 8 C A G G A
Exon 9
Exon 9
T G G G G T G A G G G C
C A A
C G A
G C T G C A
G C T G
C G
C T G G
C A
G G A
G T A
C
C G G G A
G
C T G GT G C T G
C T G C
Exon 9
Exon 8 (-12bp) C A G G A T G G G G A
G C T G
C A
G
C T G
C G
C TG G
C A
G G A GT A
C
C G G G A
G
C T G GT G C T G C T G C T G C
Exon 8 with retained intron 8 with premature termination codon CA G G
AT G
G
G
G T G
A G
G G C C A A C G
T G
A G
T G G G
G G G C C C G
AC G C C C C G
A C G
A C C C C T G
A C A G
C C G C C C
Exon 9
Exon 8 (-12bp) C A G G A T G G G G A G C T G C A
G
G
C T G C G
C T G G C A
G G A GT A
C
C G G G A
G
C T G GT G C T G C T G C T G C
Figure 1. Alternative splicing in EXON 8 of PLEC. (a) RT-PCR analysis of patient RNA isolated from cultured patient keratinocytes showed two products on a 3% agarose gel (lane 2). The larger product of 223 kb contained the retention of shortened intron 8 of 59 bp. This intron retention results in a PTC and absence of
2
Journal of Investigative Dermatology (2016), Volume
-
ska et al. KB Gostyn
Shortened Human PLEC Isoform
a
b
c
d
299-302 Human Norwegian rat Rhesus Chimpanzee Horse Zebrafish Woodpecker
PD VQDG VRA N E L Q PGAQDG VRA N E L Q PD VQDG VRA N E L Q PD VQDG VRA N E L Q PD VQDG VK A N E L Q PD VRDG VK A N E L E P E VQDG VK A N - - -
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 wild-type product and alternatively spliced product. (b), (c) Western blot using the 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 approximately 500 kDa). Quantification of plectin expression by patient keratinocytes showed 30% expression, compared with healthy control keratinocytes (b), and 10% expression by patient fibroblasts, compared with healthy control fibroblasts (c). (d) Conservation of plectin residues 299e302, which are omitted in the novel isoform. RT-PCR, reverse transcription-PCR.
This shorter mRNA transcript was also found in skin of healthy control individuals at the same level, and a quantitative 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 with healthy control keratinocytes and fibroblasts, respectively (Figure 2b and c). Reverse
transcription-PCR analysis on RNA isolated from healthy human striated muscle and myocardium samples also showed the presence of the alternatively spliced transcript (Figure 2a). 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 with pyloric atresia phenotype, whereas expression of only rodless plectin results in an EBS with muscular dystrophy phenotype (Pfendner and Uitto, 2005; Rezniczek et al., 2010; Winter and Wiche, 2013). We believe that our patient would have developed the more severe, lethal form, EBS with pyloric atresia, 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 muscular dystrophy, as her muscle symptoms are gradually worsening. This could be due to the lower amount of plectin production compared with normal control skin, that is, 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 with muscular dystrophy phenotype in the presented patient. CONFLICT OF INTEREST The authors state no conflict of interest.
= 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 3e7. Pictured to the right is the quantification of the RT-PCR of the different products. There was no difference in quantity of PLEC transcripts that arose from the 50 alternative splice site between healthy controls and patient. The slight difference in ratio between PLEC transcripts that arose from the 50 consensus splice site could be explained by nonsense-mediated mRNA decay of the consensus spliced transcript 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 blue lowercase letters, whereas the consensus splice site is marked with a blue line, and the alternative splice site with a maroon 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 maroon correspond with the RNA transcripts that are observed in the agarose gel in (a). (d) The numbers in blue and maroon correspond to (a). PCT, premature termination codon; RT-PCR, reverse transcription-PCR.
www.jidonline.org
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ska et al. KB Gostyn
Shortened Human PLEC Isoform ACKNOWLEDGMENTS We thank the patient for her cooperation in this study. We also thank Janny Zuiderveen, Gonnie Meijer, Laura van Nijen-Vos, and Daryll Eichhorn for their assistance in the lab. JB, PCvdA, and AMGP are sponsored by the Priority Medicines Rare Diseases (E-RARE) grant SpliceEB from the Netherlands Organization for Health Research and Development (ZonMw), and PCvdA also by a Clinical Fellowship grant (90715614) from ZonMW. JB, PCvdA, and AMGP are part of COST action BM1207. The Dutch Butterfly Child Foundation (Vlinderkind) also sponsored this research.
ska1,*, Katarzyna B. Gostyn 2,3 Henny Lemmink , Jeroen Bremer1,3, Hendri H. Pas1, Miranda Nijenhuis1, Peter C. van den Akker2, Richard J. Sinke2, Marcel F. Jonkman1 and Anna M.G. Pasmooij1 1
University of Groningen, University Medical Center Groningen, Department of Dermatology, Center for Blistering Diseases, Groningen, the Netherlands; and 2University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands 3
These authors contributed equally to this work.
*
Corresponding author e-mail: k.gostynska@ umcg.nl
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www.jidonline.org, and at http://dx.doi.org/10.1016/j.jid.2016.09.032.
REFERENCES Andra K, Kornacker I, Jorgl A, Zorer M, Spazierer D, Fuchs P, et al. Plectin-isoform-
4
specific rescue of hemidesmosomal defects in plectin (/) keratinocytes. J Invest Dermatol 2003;120:189e97. Bolling MC, Jongbloed JD, Boven LG, Diercks GF, Smith FJ, McLean WH, et al. Plectin mutations underlie epidermolysis bullosa simplex in 8% of patients. J Invest Dermatol 2014;134:273e6. Fuchs P, Zorer M, Rezniczek GA, Spazierer D, Oehler S, Castanon MJ, et al. Unusual 5’ transcript complexity of plectin isoforms: novel tissue-specific exons modulate actin binding activity. Hum Mol Genet 1999;8: 2461e72. Gostynska KB, Nijenhuis M, Lemmink H, Pas HH, Pasmooij AM, Lang KK, et al. Mutation in exon 1a of PLEC, leading to disruption of plectin isoform 1a, causes autosomal-recessive skin-only epidermolysis bullosa simplex. Hum Mol Genet 2015;24: 3155e62. Ketema M, Secades P, Kreft M, Nahidiazar L, Janssen H, Jalink K, et al. The rod domain is not essential for the function of plectin in maintaining tissue integrity. Mol Biol Cell 2015;26: 2402e17. Koss-Harnes D, Hoyheim B, Anton-Lamprecht I, Gjesti A, Jorgensen RS, Jahnsen FL, et al. A sitespecific plectin mutation causes dominant epidermolysis bullosa simplex Ogna: two identical de novo mutations. J Invest Dermatol 2002;118: 87e93. Koster J, Geerts D, Favre B, Borradori L, Sonnenberg A. Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly. J Cell Sci 2003;116(Pt 2):387e99. Litjens SH, Koster J, Kuikman I, van Wilpe S, de Pereda JM, Sonnenberg A. Specificity of binding of the plectin actin-binding domain to
Journal of Investigative Dermatology (2016), Volume
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beta4 integrin. 4039e50.
Mol
Biol
Cell
2003;14:
McLean WH, Pulkkinen L, Smith FJ, Rugg EL, Lane EB, Bullrich F, et al. Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization. Genes Dev 1996;10:1724e35. Natsuga K, Nishie W, Akiyama M, Nakamura H, Shinkuma S, McMillan JR, et al. Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex. Hum Mutat 2010;31:308e16. Pfendner E, Uitto J. Plectin gene mutations can cause epidermolysis bullosa with pyloric atresia. J Invest Dermatol 2005;124:111e5. Rezniczek GA, Abrahamsberg C, Fuchs P, Spazierer D, Wiche G. Plectin 5’-transcript diversity: short alternative sequences determine stability of gene products, initiation of translation and subcellular localization of isoforms. Hum Mol Genet 2003;12: 3181e94. Rezniczek GA, Walko G, Wiche G. Plectin gene defects lead to various forms of epidermolysis bullosa simplex. Dermatol Clin 2010;28: 33e41. Sikkema-Raddatz B, Johansson LF, de Boer EN, Almomani R, Boven LG, van den Berg MP, et al. Targeted next-generation sequencing can replace Sanger sequencing in clinical diagnostics. Hum Mutat 2013;34: 1035e42. Smith FJ, Eady RA, Leigh IM, McMillan JR, Rugg EL, Kelsell DP, et al. Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat Genet 1996;13: 450e7. Winter L, Wiche G. The many faces of plectin and plectinopathies: pathology and mechanisms. Acta Neuropathol 2013;125:77e93.
A PLEC Isoform Identified in Skin, Muscle, and Heart Journal of Investigative Dermatology (2016) -, -e-; doi:10.1016/j.jid.2016.09.032
TO THE EDITOR 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 a6b4 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 tissuespecific 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 (Ketema et al., 2015; Winter and Wiche, 2013). 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 (Natsuga et al., 2010) or muscular dystrophy (Smith et al., 1996). Here, we describe an alternative PLEC isoform expressed in skin, muscle, and heart, which is the result of a thus far unreported alternative splicing event in exon 8. The proband (IV-4, EB 210-01, Supplementary Figure S1 online) presented at 17 years of age with complaints of acral skin blistering since the age of 2 years. The proband gave written and informed consent for publication of clinical photographs and description. No institutional approval
was required as all performed methods were a part of routine clinical diagnostics. She was the daughter of consanguineous Moroccan parents. Acral blistering, subtle nail pitting of the hands, focal plantar hyperkeratosis, onychokeratosis, and onycholysis were observed (Supplementary Figure S2 online). 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 (Supplementary Figure S1). After her brother’s death, the proband and her older sister (IV-3) underwent cardiologic screening, which had disclosed a cardiomyopathy. Immunofluorescence 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 (Supplementary Figure S3a and b online). Transmission electron microscopy further visualized pseudojunctional cleavage with the plasma membrane and the lamina densa in the blister floor (Supplementary Figure S3c and d). Taken together, the data suggested a
Abbreviation: EBS, epidermolysis bullosa simplex Accepted manuscript published online 18 October 2016; corrected proof published online XXX XXXX ª 2016 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
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 (Supplementary Table S1 online) (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.906þ19_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 reverse transcription-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, 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 wild type. 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 299e302 when compared with wildtype plectin. Although these amino acids 299e302 are conserved, they are just outside the actin-binding domain of plectin (Litjens et al., 2003) (Figure 2d). www.jidonline.org
1
ska et al. KB Gostyn
Shortened Human PLEC Isoform Total mRNA template (arbitrary units)
a
2 1 1
2
35.000 30.000 25.000 20.000 15.000 10.000 5.000 0
Control Patient (n=5) Consensus splicing
Control Patient (n=5) Alternative splicing
b AGGATGGG GTGAGGGCCAAC gtgagtggggggcccggagggcagggggcttgggcctggacgccccgacgacccctgacagccgcccgtgcctgcccgcag GAGCTGCAGCT Intron 8 Exon 9 Exon 8
c
Healthy individual 1
standard splicing Intron 8 (81bp)
Exon 8 1
Exon 9
alternate splicing Intron 8 (81bp)
Exon 8
Exon 9
Patient IV-4 2
PTC
standard splicing Exon 8
2
alternate splicing Intron 8 (59bp)
Patient IV-4 (2)
Patient IV-4 (2)
Healthy individual (1) Healthy individual (1)
Exon 8
d
Exon 9
Intron 8 (59bp)
Exon 8 C A G G A
Exon 9
Exon 9
T G G G G T G A G G G C
C A A
C G A
G C T G C A
G C T G
C G
C T G G
C A
G G A
G T A
C
C G G G A
G
C T G GT G C T G
C T G C
Exon 9
Exon 8 (-12bp) C A G G A T G G G G A
G C T G
C A
G
C T G
C G
C TG G
C A
G G A GT A
C
C G G G A
G
C T G GT G C T G C T G C T G C
Exon 8 with retained intron 8 with premature termination codon CA G G
AT G
G
G
G T G
A G
G G C C A A C G
T G
A G
T G G G
G G G C C C G
AC G C C C C G
A C G
A C C C C T G
A C A G
C C G C C C
Exon 9
Exon 8 (-12bp) C A G G A T G G G G A G C T G C A
G
G
C T G C G
C T G G C A
G G A GT A
C
C G G G A
G
C T G GT G C T G C T G C T G C
Figure 1. Alternative splicing in EXON 8 of PLEC. (a) RT-PCR analysis of patient RNA isolated from cultured patient keratinocytes showed two products on a 3% agarose gel (lane 2). The larger product of 223 kb contained the retention of shortened intron 8 of 59 bp. This intron retention results in a PTC and absence of
2
Journal of Investigative Dermatology (2016), Volume
-
ska et al. KB Gostyn
Shortened Human PLEC Isoform
a
b
c
d
299-302 Human Norwegian rat Rhesus Chimpanzee Horse Zebrafish Woodpecker
PD VQDG VRA N E L Q PGAQDG VRA N E L Q PD VQDG VRA N E L Q PD VQDG VRA N E L Q PD VQDG VK A N E L Q PD VRDG VK A N E L E P E VQDG VK A N - - -
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 wild-type product and alternatively spliced product. (b), (c) Western blot using the 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 approximately 500 kDa). Quantification of plectin expression by patient keratinocytes showed 30% expression, compared with healthy control keratinocytes (b), and 10% expression by patient fibroblasts, compared with healthy control fibroblasts (c). (d) Conservation of plectin residues 299e302, which are omitted in the novel isoform. RT-PCR, reverse transcription-PCR.
This shorter mRNA transcript was also found in skin of healthy control individuals at the same level, and a quantitative 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 with healthy control keratinocytes and fibroblasts, respectively (Figure 2b and c). Reverse
transcription-PCR analysis on RNA isolated from healthy human striated muscle and myocardium samples also showed the presence of the alternatively spliced transcript (Figure 2a). 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 with pyloric atresia phenotype, whereas expression of only rodless plectin results in an EBS with muscular dystrophy phenotype (Pfendner and Uitto, 2005; Rezniczek et al., 2010; Winter and Wiche, 2013). We believe that our patient would have developed the more severe, lethal form, EBS with pyloric atresia, 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 muscular dystrophy, as her muscle symptoms are gradually worsening. This could be due to the lower amount of plectin production compared with normal control skin, that is, 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 with muscular dystrophy phenotype in the presented patient. CONFLICT OF INTEREST The authors state no conflict of interest.
= 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 3e7. Pictured to the right is the quantification of the RT-PCR of the different products. There was no difference in quantity of PLEC transcripts that arose from the 50 alternative splice site between healthy controls and patient. The slight difference in ratio between PLEC transcripts that arose from the 50 consensus splice site could be explained by nonsense-mediated mRNA decay of the consensus spliced transcript 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 blue lowercase letters, whereas the consensus splice site is marked with a blue line, and the alternative splice site with a maroon 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 maroon correspond with the RNA transcripts that are observed in the agarose gel in (a). (d) The numbers in blue and maroon correspond to (a). PCT, premature termination codon; RT-PCR, reverse transcription-PCR.
www.jidonline.org
3
ska et al. KB Gostyn
Shortened Human PLEC Isoform ACKNOWLEDGMENTS We thank the patient for her cooperation in this study. We also thank Janny Zuiderveen, Gonnie Meijer, Laura van Nijen-Vos, and Daryll Eichhorn for their assistance in the lab. JB, PCvdA, and AMGP are sponsored by the Priority Medicines Rare Diseases (E-RARE) grant SpliceEB from the Netherlands Organization for Health Research and Development (ZonMw), and PCvdA also by a Clinical Fellowship grant (90715614) from ZonMW. JB, PCvdA, and AMGP are part of COST action BM1207. The Dutch Butterfly Child Foundation (Vlinderkind) also sponsored this research.
ska1,*, Katarzyna B. Gostyn 2,3 Henny Lemmink , Jeroen Bremer1,3, Hendri H. Pas1, Miranda Nijenhuis1, Peter C. van den Akker2, Richard J. Sinke2, Marcel F. Jonkman1 and Anna M.G. Pasmooij1 1
University of Groningen, University Medical Center Groningen, Department of Dermatology, Center for Blistering Diseases, Groningen, the Netherlands; and 2University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands 3
These authors contributed equally to this work.
*
Corresponding author e-mail: k.gostynska@ umcg.nl
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www.jidonline.org, and at http://dx.doi.org/10.1016/j.jid.2016.09.032.
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