University of Groningen

Epidermolysis bullosa simplexBolling, Maria Caroline

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3

Plectin mutations in basal epidermolysis bullosa simplex with

wild-type KRT5 and KRT14 genes

MC Bolling1, JDH Jongbloed2, LGH Boven2, GFH. Diercks3, FJD Smith4, WHI McLean4, MF Jonkman1

Departments of 1Dermatology, 2Genetics, and 3Pathology, University Medical Center Groningen, Groningen, The Netherlands;

and 4Epithelial Genetics Group, Division of Molecular Medicine, Colleges of Life Sciences and Medicine, Dentistry and Nursing,

University of Dundee, Dundee, United Kingdom

Submitted

82

Chapter 3

Abstract

Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis characterized

by cytoskeleton fragility of basal keratinocytes caused by mutations in the KRT5 and KRT14

genes, encoding the basal epidermal keratins. However, in ~25% of individuals in an EBS case

series from the Netherlands (n=65), no mutation was found in either gene. One autosomal

dominant mutation in another gene, PLEC1, has been associated with non-syndromic EBS, a

phenotype called EBS-Ogna. PLEC1 encodes the cytoskeleton linker protein plectin, which

in skin anchors basal keratins to the hemidesmosomal plaque. In this study, PLEC1 mutation

analysis was performed in 16 EBS probands in which KRT5 and KRT14 mutations had been

excluded. In four of these probands, a heterozygous plectin missense mutation was found (25%

of cases analyzed; 6% of total EBS cases). Two probands carried the same plectin mutation as

the original EBS-Ogna families: p.Arg2000Trp. The clinical phenotype is characterized by acral

blistering, haemorrhagic elements on the hand dorsum, pretibial atrophy, and thickened,

discoloured toenails. Immunofluorescence microscopy revealed diminished plectin expression.

Ultrastructural analysis showed cleavage just above the inner dense plaque of variably

hypoplastic hemidesmosomes. This study demonstrates that PLEC1 is the third candidate gene

to screen for mutations in EBS.

83

Plectin mutations in basal EBS

Introduction

Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis characterized by an

intraepidermal split through the cytoplasm of the basal keratinocytes. Three main subtypes

exist based on onset, severity and localization of blistering: EBS-localized (EBS-loc) with

blistering confined to hands and feet from infancy, EBS-generalized, non-Dowling Meara (EBS-

gen) with early onset of generalized blistering, and EBS Dowling Meara (EBS-DM) with severe

congenital circinate (‘herpetiform’) blistering often involving mucosal surfaces, development of

palmoplantar keratoderma and the characteristic clumping of keratin filaments in skin samples

visualised by electron microscopic (EM) analysis.1 In the majority of cases EBS is caused by

autosomal dominant mutations in the KRT5 and KRT14 genes encoding the basal epidermal

keratins 5 (K5) and 14 (K14), respectively.2-4

Mutation analysis of KRT5 and KRT14 in 64 biopsy-confirmed EBS patients in the

Netherlands revealed that in 25% of cases no mutations could be identified in these genes

(manuscript under submission). A similar percentage of EBS cases with wild-type KRT5 and

KRT14 genes was reported for the EBS population in the UK.5 Single unique cases of EBS caused

by recessive mutations in genes encoding the hemidesmosomal proteins type XVII collagen

(BP180) and integrin β4 (β4) have been reported.6-8 Moreover, in EBS patients of a large

Norwegian kindred from Ogna and an unrelated German kindred a heterozygous missense

mutation (p.Arg2000Trp) was found in the plectin gene (EBS-Ogna, MIM#131950).9-11

Plectin is a widely expressed and versatile cytoskeletal linker protein. Multiple plectin

isoforms exist that result from alternative splicing of the PLEC1 N-terminal region.12-14 In addition,

evidence is found for a rodless plectin isoform resulting from out-splicing of exon 31. These

different plectin isoforms are expressed in a tissue-specific way in a wide variety of tissues,

including skin, striated muscle and heart, where they play a pivotal role in interactions between

different cytoskeletal systems and the anchorage of these systems to cell-cell and cell-matrix

junctions. In skin, plectin is localized in hemidesmosomes, where it anchors the keratin filaments

to the cytoplasmatic domains of β4 and BP180.15 Plectin also is present in desmosomes where it

is colocalized with desmoplakin.16 Plectin knockout mice die few days after birth showing skin,

skeletal muscle and myocardial pathology.17

In humans, PLEC1 mutations are associated with intraepidermal skin fragility, and

except for EBS-Ogna, they are all autosomal recessive. In general, when mutations result in loss

of the full-length plectin isoforms they cause EBS with muscular dystrophy.18 When the rodless

plectin isoform is affected as well, the responsible mutations are associated with early lethal

phenotypes, like EBS with pyloric atresia.19, 20

In the present study we investigated whether PLEC1 mutations are involved in EBS in

patients with normal KRT5 and KRT14 genes. The results indicate that in addition to KRT5 and

KRT14 mutations, the number of PLEC1 mutations in EBS is significant.

84

Chapter 3

Material and methods

Patients

In 16 of 65 (25%) unrelated probands with a diagnosis of EBS based on clinical features and biopsy

findings that were registered in our Dermatology clinic between 1989 and 2008 no mutations

could be found in the KRT5 and KRT14 genes using regular mutation analysis techniques.21, 22

Two cases with pseudojunctional EBS due to recessive mutations in COL17A17 and ITGB48 were

excluded. All patients gave their informed consent and the study was performed according to

the Declaration of Helsinki protocols.

Skin and blood samples

For immunofluorescence microscopy, two 4-mm skin biopsies were taken from each patient,

one from a blister and one from healthy looking skin from the inner aspect upper arm, and snap-

frozen in liquid nitrogen. For EM two 2-mm skin biopsies were taken from the same locations,

and fixed in glutaraldehyde 2%. EM biopsies of proband 3 were not available. DNA was extracted

from peripheral blood lymphocytes from the probands and available family members using 6 M

NaCl and chloroform as previously described.23

Exclusion of KRT5 and KRT14 mutations

PCR-based amplification and direct sequencing of all exons and adjacent intronic sequences

of the KRT5 and KRT14 genes had been performed in all 16 probands.21, 22 To search for large

in-frame deletions or duplications that might be missed by amplification of single exons,

gDNA of the 16 probands was subjected to Long Range PCR (LR-PCR) using the Expand Long

Template PCR system (Roche Applied Science, Almere, Netherlands) with forward primers in

the 5’ region of the first exon of KRT5 and KRT14, and reverse primers in the 3’ region of the

last exon of these genes. This was predicted to result in PCR products of 5481 bp (KRT5) and

4470 bp (KRT14), respectively, using control gDNA as template (primer sequences available

on request). The lengths of these fragments and PCR fragments resulting from amplification

using patients’ gDNA as template were analysed on a 1% agarose gel. In addition, RNA of

the 16 probands was analyzed for deletions or insertions. RNA was isolated from four 10 µm

cryosections of nonlesional skin samples of the probands according to the Absolutely RNA

Microprep Kit protocol (Stratagene Europe, Heidelberg, Germany). cDNA was subsequently

synthesized according to the manufacturer’s protocol (Invitrogen, Breda, The Netherlands). PCR

amplification was performed on cDNA with forward primers annealing to sequences located in

exon 1 and reverse primers annealing to sequences located in the last exons of KRT5 and KRT14,

predicted to result in PCR fragment lengths of 1726 bp (KRT5) and 1336 bp (KRT14) using control

cDNA as template (primer sequences available on request). The lengths of these fragments and

the PCR fragments amplified using patients’ cDNA as template were analysed on a 1% agarose

gels.

85

Plectin mutations in basal EBS

PLEC1 mutation analysis

PCR amplification of all exons and adjacent intronic sequences of PLEC1 (GenBank accession

number NM_000436.3) was performed using primers located in the flanking introns. Primers

resulting in overlapping PCR products were used for the large exons 31 and 32 (information

about primers and amplification conditions is available on request). DNA from at least 150

healthy individuals was used to screen for the presence/absence of the identified PLEC1

mutations.

Bioinformatics

The SNP database of NCBI was searched to exclude that detected amino acid substitutions in

the plectin gene were known as polymorphisms.24 To analyze the evolutionary conservation

of the mutated amino acids, protein sequence homologues to the human plectin rod domain

and C-terminal PRDs were gathered with NCBI-BLAST. Comparative sequence analysis (multiple

sequence analysis [MSA]) was performed using ClustalW2 with a Java viewer (Jalview).

Immunofluorescence and electron microscopy

For immunofluorescence microscopy 4 mm cryostat sections were cut from snap-frozen skin

biopsies of the patients and processed as previously described.25 The following antibodies

were used: rabbit BL18 against K5 and mouse LL001 against K14 (Prof E. B. Lane); mouse HD121

(Dr K. Owaribe) and mouse 10F6 against the plectin rod domain (Santa Cruz Biotechnology,

Santa Cruz, CA); mouse 1A8c (endodomain) and mouse 1D1 (ectodomain) against BP180 (Dr

K. Owaribe, Nagoya, Japan); 58XB4 (ectodomain) and clone 7 (endodomain) against β4 (Dr A.

Sonnenberg). As a secondary step Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes

Europe, Leiden, the Netherlands) was used in combination with primary mouse antibodies.

FITC labelled goat anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL, USA)

was used in combination with primary rabbit antibodies and FITC labelled rabbit anti-goat IgG

(Dako, Glostrup, Denmark) in combination with primary goat antibodies. Nuclei were stained

with bisbenzimide. EM was performed as previously described 25.

Results

No mutations in KRT5 or KRT14

No mutations were found by amplification and direct sequencing of all exons including the exon-

intron borders of the KRT5 and KRT14 genes of genomic DNA (gDNA) of 16 probands with EBS

out of a total number of 65 unrelated EBS probands (25%). Additional KRT5 and KRT14 long range

PCR analysis on gDNA and cDNA samples derived from patient keratinocytes and subsequent

analysis of fragment lengths did not reveal large deletions or insertions. Immunofluorescence

antigen mapping showed normal amounts of K5 and K14 in skin samples of all probands (data

not shown). In two of the 16 probands keratin clumping was demonstrated fitting a diagnosis of

86

Chapter 3

EBS-DM. In two others EBS-gen non-DM was diagnosed, and in 12 probands the phenotype was

EBS-loc (for details on the clinical features see table S1).

Table S1. Details on the clinical features of EBS probands without KRT5 or KRT14 mutations

Family (*)

EBS subtype

Inheri-tance

Origin Onset Blistering Other

1(EB184)

Loc AD Dutch < 3 y Hands, feet, lower legs; blood blebs and erosions; seasonal variation

Nail dystrophy

2 (EB149)

Loc Sp Dutch < 1 y Hands, feet, lower legs; blood blebs and erosions; seasonal variation

Nail dystrophy

3(EB146)

Loc AD Iran < 1 y Hands, feet, lower legs; seasonal variation

Nail dystrophy

4(EB203)

Loc AD Dutch 1-2 y Hands, feet, lower legs; seasonal variation

Nail dystrophy

5(EB013)

Loc Sp Dutch < 1 y Hands, feet, very mild; seasonal variation

6(EB003)

Gen AD Dutch Birth Generalized, also mucosal Aplasia cutis

7(EB085)

DM Sp Dutch Birth Generalized, also mucosal, grouped, circinary

Palmoplantar keratoderma, erosions below nailplate; clumping in EM

8(EB087)

Loc AD Dutch < 1 y Hands and feet; seasonal variation

9(EB092)

DM AD Dutch Birth Generalized, also mucosal, grouped, circinary; marked improvement after puberty

Aplasia cutis; palmoplantar keratoderma, erosions below nail plate; clumping in EM

10(EB118)

Gen Sp Dutch < 1 y Generalized Palmoplantar keratoderma

11(EB131)

Loc Sp Middle-East

2 d Hands and feet; seasonal variation

12(EB104)

Loc AD Dutch ~6 y Hands and feet; seasonal variation Plantar keratoderma

13(EB193)

Loc AD Dutch 2-6 y Hands and feet; seasonal variation

14(EB143)

Loc Sp Dutch 6 y Hands and feet; blood blebs; seasonal variation

15(EB123)

Loc Sp Dutch ~4 y Feet; seasonal variation

16(EB185)

Loc Sp Dutch < 1 y Hands and feet

AD, autosomal dominant; d, day; DM, Dowling-Meara; EM, electron microscopy; gen, generalized non-DM; loc, localized; Sp, sporadic; y, year(*) Patient-code EB-database Center for Blistering Disease, Groningen, The Netherlands

87

Plectin mutations in basal EBS

Table 1. Characteristics of the PLEC1 mutations found in this study and associated IF findings in patient skin biopsies.

Proband Mutation c. § Mutation p.# Domain Conservation $ Immunofluorescence analysis skin

1 (EB184) 5998C>T(CGG>TGG)

Arg2000Trp Coiled-coil rod domain m, d, zf, fr HD121 , 10F6 absent in basal epidermal layer

2 (EB149) 5998C>T (CGG>TGG)

Arg2000Trp Coiled-coil rod domain m, d, zf, fr HD121 , 10F6 absent in basal epidermal layer

3 (EB146) 8668A>T(ACG<TCG)

Thr2890Ser Plakin repeat domain 1 m, d, zf HD121~ normal, 10F6 slightly panepidermal

4 (EB203) 10579C>T(CGC>TGC)

Arg3527Cys Plakin repeat domain 3 m, d HD121~ normal, 10F6 slightly panepidermal

§ numbering according to GenBank NM_000445.3 with 1 being the adenine from the ATG startcodon# GenBank NP_000436.2 (isoform 1c)$ m, mouse; d, dog; zf, zebrafish; fr, frog

PLEC1 mutations

Mutation analysis in the 16 EBS probands with wild-type KRT5 and KRT14 alleles revealed

heterozygous single nucleotide changes in PLEC1 in four probands (25%) (figure 1). The

mutations are summarized in table 1. The mutations were excluded in at least 150 control gDNA

samples. Of note, unfortunately this result is of less value in the case of the Iranian proband (#3),

as these control samples were derived from Dutch individuals. The amino acid changes were not

listed as known polymorphisms in the NCBI SNP database and segregated with the phenotype

in the families.

Probands 1 and 2 carried the same heterozygous PLEC1 mutation as found in the

original EBS-Ogna families: c.5998C>T, p.Arg2000Trp. The encoding trinucleotide of the arginine

residue contains a CpG dinucleotide in the PLEC1 gene. The p.Arg2000 residue resides in the

rod domain of the plectin protein and is well conserved among species (figure S2). The affected

mother of the proband 1 was deceased and no DNA was available to confirm transmission of

the mutation. Proband 2 was the only affected person in the family. Neither parents carried the

mutation suggesting that this was a de novo event.

In probands 3 and 4, heterozygous missense mutations were found within the

C-terminal plakin repeat domains (PRDs) – the part of the protein implicated in keratin

interaction. Proband 3 carried the mutation c.8668A>T, p.Thr2890Ser. Mutation p.Thr2890 is

located in PRD1 in the plectin C-terminus and is well conserved among species (figure S2). An

affected brother carried the mutation whereas an unaffected son did not carry the mutation. In

proband 4 a heterozygous missense mutation was detected: c.10597C>T, p.Arg3527Cys. This

mutation was found in his affected mother as well. The unaffected siblings and father did not

carry the mutation. Residue p.Arg3527 resides in the third PRD in the C-terminus of plectin and

the arginine at this position is conserved in mammals (figure S2).

88

Chapter 3

Figure 1. PLEC1 mutations in EBS. (a) Mutation p.Arg2000Trp (c.5998C>T) in exon 31. (b) Mutation p.Thr2890Ser (c.8668A>T) in exon 32. (c) Mutation p.Arg3527Cys (c.10579C>T) in exon 32. (d) Schematic representation of the plectin protein with the mutations depicted below the protein. GenBank accession number NM_000436.3.

Clinical features of patients with PLEC1 mutations

Here, we refer to the EBS patients with plectin missense mutations as ‘plectin-EBS’. Proband 1

with mutation p.Arg2000Trp was a 45-year old man with blistering and erosions since early

childhood, mainly affecting hands, feet and lower legs (figure 2a). The blisters were often filled

with blood and on the lower legs healed with atrophic scarring. Blistering improved during

adolescence but worsened again in adulthood, probably because of severe insufficiency of the

lower leg superficial veins. All toenails and the fingernails of digits I and II of the left hand were

thickened and discoloured. Hair and teeth were normal. The deceased mother of the proband

had been similarly affected and the father was unknown. A sister and brother were unaffected.

Proband 2 with mutation p.Arg2000Trp was a 16 year old boy with blistering of mainly

hands, feet and lower legs from infancy (figure 2b). On the dorsal sides of the hands small

erosions and blood blebs were often present. The blisters easily became erosive and healed

slowly. On the lower legs the blistering healed with atrophic scarring. The nails of digits I on

both feet showed distal onycholysis with thickening and discoloration. Hair and teeth were

unaffected. The proband was the only affected person in the family suggesting a de novo event.

Both non-consanguinous parents and two siblings were unaffected.

Proband 3 with mutation p.Thr2890Ser was a 46 year-old male of Iranian descent

who displayed blistering and erosions on hands and feet since early childhood (figure 2c). The

blistering was reported to be more severe in the summer months. The nails of the great toes

showed thickening. Hair and teeth were normal. His brother and one son showed similar skin

fragility, another son was unaffected. The history of the parents was unclear.

a

b

c

Arg2000Trp

Arg3527Cys

WT

WT

WT

NH2 COOHCH1- CH2

2-8 9-30 31 32Exon nr

CH1- CH2actin binding domain consisting of two calponin homology (CH) domains

globular plakin domain with spectrin repeats

central coiled-coil rod domain

c-terminal plakin repeats with intermediate filament binding motif

R2000W T2890S R3527C

Thr3934Met

d

C>T

A>T

C>T

89

Plectin mutations in basal EBS

Proband 4 with mutations p.Arg3527Cys was a 44-year old man with blistering,

erosions and small blood blebs on hands and feet from the age of 2 years that worsened in the

summer months (figure 2d). The toenails were thickened and discoloured. Mucosae, hair and

teeth were unaffected. His mother was similarly affected. His father, two brothers and two sons

were unaffected.

Cardiological and neurological examination excluded cardiomyopathy and muscular

dystrophy in all probands.

Figure 2. (a) Blood filled (open arrow head) as well as serous (arrow) blisters on the right foot of patient EB184 (family 1). On the frontal lower legs, atrophic scarring can be seen. Erosions on the dorsal side of the right hand (open arrow heads) and thickening and discolourisation of all toenails can be seen. (b) Old blister on the heel of patient EB149 (family 2) and erosions on the dorsal side of the right hand (open arrowhead). Note the atrophic scar on digitum II. Thickening and distal onycholysis on the toenails of digits I. (c) Blistering on plantar skin and digitum I on the right hand

(arrows). Thickening of the toenails of digits I in patient EB146 (family 3). (d) Blister (arrow) on plantar skin of EB203 (family 4) with a detail of a vesicle on the lateral aspect of the fourth toe. Toenails of digits I were thickened and showed an increased distal curve.

Abnormal and normal hemidesmosomes

EM analysis of nonlesional skin of the probands revealed normal hemidesmosomes, as well as

hemidesmosomes with thin and/or interrupted outer plaques and impaired insertion of keratin

filaments into the hemidesmosomal plaque (figure 3a).

In lesional biopsies an intraepidermal split level just above the hemidesmosomal

plaque was observed showing a plasma membrane and foci of keratin filaments on the blister

floor (figure 3b). In some regions, we found lamina densa on the blister floor mimicking a focal

junctional split through the lamina lucida (‘pseudojunctional split’). None of the skin samples

showed evidence of keratin aggregation.

a

b

c

d

Arg2000Trp

Proband 3Thr2890Ser

Proband 4Arg3527Cys

Proband 1

Proband 2

^

^

^

^^

^

90

Chapter 3

Figure 3. Impaired keratin filament insertion and low cytoplasmatic split in basal keratinocytes. (a) Hemidesmosomes are shown with a normal plaque (arrow), with a discontinuous outer plaque (double arrow), or with a hypoplastic plaque (open arrowhead) (b) Blister (asterisk) of proband 1 reveals a low intracytoplasmic split above the plasmamembrane (arrow) of a basal keratinocyte. The blister floor also contains keratin cell remnants (double arrow), and areas of naked lamina densa (open arrowhead) due to secondary loss of debris, thus mimicking a junctional split (‘pseudojunctional’ split). Scale bar represents 500 nm.

Altered plectin staining

Immunofluorescence antigen mapping revealed a varying level of blistering with some blisters

having a cytoplasmic plane of cleavage as shown by overt keratin staining on the blister floor

(EBS), while others showed a plane just above the hemidesmosome with a minimal amount of

keratin speckles on the blister floor, suggesting low basal cell cleavage.

In control skin, plectin staining with antibody HD121 (figure 4a) showed linear staining

along the epidermal basement membrane zone (EBMZ) and a subtle intercellular substance

(ICS) staining in all layers of the epidermis. However, in skin samples of probands 1 and 2

(mutation Arg2000Trp) HD121 staining was slightly reduced along the EBMZ and the epidermal

ICS staining was absent (figure 4a). Skin of probands 3 (Thr2890Ser) and 4 (Arg3527Cys) showed

normal HD121 staining. In control skin anti-plectin antibody 10F6 (figure 4b) stained the ICS

more prominently in all layers of the control epidermis than HD121, while the EBMZ staining

was less pronounced. In marked contrast, 10F6 staining at the EBMZ and of the basal cell layer

was markedly reduced to absent in skin of probands 1 and 2 and slightly reduced overall in

probands 3 and 4. The 10F6 plectin epitope was normal expressed in BMZ of dermal vessels.

Expression of other hemidesmosomal components BP180 and β4 along the EBMZ was

unaltered in all probands (data not shown).

*

<

a

b

<

91

Plectin mutations in basal EBS

Figure 4. (a) Plectin staining with antibody HD121 is reduced at the EBMZ in skin samples of proband 1 and 2, and normal in proband 3. The slight panepidermal ICS staining (arrow) observed in control skin samples is absent in skin of proband 1, 2 and 3. Note the staining of HD121 in blister roof and floor of proband 1 (*), and only in blister floor of proband 3 (#). (b) Plectin staining with antibody 10F6 is slightly reduced in the suprabasal epidermal layers and markedly reduced to absent in the basal epidermal layer and EBMZ in lesional and healthy skin of probands 1 and 2 (arrows) compared to control skin. The 10F6 staining is preserved in skin of proband 3. Note the preserved 10F6 staining of dermal vessels skin samples of all probands. Scale bar represents 50 um.

Discussion

In this study, we show that plectin mutations underlie an EBS phenotype in four of 16 probands

(25%) with wild-type KRT5 or KRT14 alleles in a total population of 65 EBS probands (6%).

The results of the present study indicate that dominant plectin mutations in general, and

the original p.Arg2000Trp EBS-Ogna mutation in particular, are not as rare in EBS as initially

believed, by adding two unrelated Dutch EBS patients carrying the p.Arg2000Trp and three

other patients carrying novel dominant plectin missense mutations associated with EBS.11 The

mutations segregated with the phenotype in the family as far as the respective families could

be investigated, were not known as SNPs, and were absent in >150 control DNA samples, thus

strongly indicating pathogenicity.

lesi

onal

ski

n pr

oban

d 1

HD121 10F6 he

alth

y sk

in p

roba

nd 2

cont

rol s

kin

lesi

onal

ski

n pr

oban

d 3

a b

*

#

#

*

92

Chapter 3

In hemidesmosomes, plectin is an important anchoring protein for the basal keratins

to β4 as was indicated by in vitro studies, by the findings in plectin knockout mice and in humans

with plectin mutations.14, 17, 18, 26 Plectin is believed to function as a parallel in-register homodimer

and/or heterodimer with other plectin isoforms.27-29 The dimerization mainly takes place

through the central rod domain, which has a highly α-helical structure favouring a coiled-coil

formation. In vitro studies have shown that presence of the plectin rod domain highly improved

hemidesmosome stability.14 Highly disruptive amino acid substitutions in the rod domain,

like the Arg>Trp substitutions at position 2000 (arginine is large, basic and positively charged,

tryptophan is hydrophobic and neutral), are likely to affect the heptad structure of the α-helical

structure. This will result in a dominant negative effect on the capability of plectin to dimerize

and therefore impair hemidesmosome stability, leading to the mild blistering phenotype

observed in patients carrying these mutations.

The phenotype in all plectin-EBS patients was of that of the localized type and four of

the five probands showed thickening of the big toenails. Other notable clinical features were the

tendency to develop small hemorrhagic blebs and erosions upon trauma on the dorsal aspects

of the hands and on the lower legs with atrophic scarring of pretibial skin. These features were

also reported in the EBS-Ogna patients.9 Ultrastructurally, we observed thin and/or fragmented

hemidesmosomal plaques with impaired keratin filament insertion. Immunofluorescence staining

with anti-plectin antibody 10F6 in skin of probands 1 and 2 carrying the rod domain mutation

Arg2000Trp showed absence of basal layer staining and a reduction along the basement membrane,

similar to the previously reported EBS-Ogna patients. However, this was not a consistent feature, as

10F6 staining was only slightly diminished panepidermal in probands 3 and 4 with the C-terminal

mutations.10, 11 Monoclonal antibody 10F6 binds to an epitope within the plectin rod domain.30

There are several possible explanations for the altered staining observed with 10F6 in skin of

patients carrying mutation p.Arg2000Trp. These include impaired binding of the antibody due

to the missense mutation, by either direct or indirect alteration of the protein tertiary structure;

or, protein degradation due to impaired protein functioning and/or positioning. Why the basal

layer shows reduced 10F6 reactivity, while the suprabasal layers are unaffected, is difficult to

understand. One explanation may be the expression of alternatively spliced plectin isoforms

differing in their rod domain composition during epidermal differentiation. On the other hand,

dimerization of plectin molecules may be a necessity for incorporation in cell junction structures in

the basal epidermal layer, while this is less important in suprabasal cells. Consequently, a missense

mutation impairing dimer formation would lead to reduced staining particularly in the basal layer.

We speculate that the missense mutation in the rod domain has a dominant-negative effect on

dimer formation, thereby leading to impaired hemidesmosomal inner dense plaque stability and

keratin anchorage. Of note, in patients with EBS due to keratin mutations, hemidesmosomes have

a normal appearance and plectin immunofluorescence staining is also normal. Therefore, reduced

10F6 basal cell staining might provide a useful diagnostic tool to predict plectin rod domain

mutations in EBS. However, additional studies are necessary to confirm this hypothesis.

93

Plectin mutations in basal EBS

The full length isoform contains an N-terminal actin-binding domain followed by nine

spectrin repeats, a central coiled-coil rod domain, six PRDs and IF-binding sites in its C-terminus,

as shown in figure 1f.31 The N-terminus interacts with a wide variety of proteins among which

actin, the hemidesmosomal proteins β4 and BP180, the nuclear envelope protein nesprin-3α,

and several muscle proteins.32-34 35, 36 Mutations p.Arg3527Cys and p.Thr2890Ser are located

outside the rod domain in the C-terminus of plectin, in PRD3 and PRD1, respectively (figure

1f ). Proband 4 carries the missense mutation p.Ag3527Cys on the maternal allele. His affected

mother also carried this mutation and two unaffected siblings and the father were negative

for this mutation, as were 150 control DNA samples. This substitution predicts considerable

alterations to protein folding. The critical keratin binding motif of plectin, resides in PRD5-

PRD6.37, 38 Mutations p.Arg3527Cys and p.Thr2890Ser do not directly affect the known IF binding

site. However, all PRDs are believed to be tightly packed to provide structural rigidity.39 The

C-terminal mutations might affect this close interaction thereby weakening the overall stability

of the plectin C-terminus and thus interfere with IF binding. In addition, a phosphorylation

site and binding sites for several cell signalling proteins have been identified in the plectin

C-terminus.40, 41 Threonine residues are often involved in phosphorylation. The C-terminal

plectin mutations might interfere with phosphorylation or the binding of regulatory proteins

that influence the affinity of plectin for IFs. Moreover, it has been proposed that the plectin

protein might contain additional keratin binding sites, which might be affected by the

C-terminal mutations.37, 38 Alternatively or perhaps additionally, these mutations could affect

plectin-β4 interactions, as plectin has been reported to contain accessory β4-binding sites in its

C-terminus in addition to the essential N-terminal binding sites.42 By affecting plectin binding to

β4 in the hemidesmosome, the C-terminal mutations might render the structure less stable and

less efficient in anchoring the keratin cytoskeleton. Additional, transfection studies or mouse

models may provide insight in the pathogenesis in future.

In conclusion, we showed that in four of 16 (25%) probands with wild-type KRT5 and

KRT14 genes dominant PLEC1 missense mutations underlie non-syndromic EBS, making PLEC1 a

major candidate gene to screen for mutations in EBS patients without KRT5 and KRT14 mutations.

The previously reported EBS-Ogna p.Arg2000Trp mutation was found in two additional unrelated

Dutch probands indicating that the Arg2000 residue might be a mutation hotspot. Important

clinical clues to diagnosis are haemorrhagic blisters, pretibial atrophy, and thickened toenails.

Ultrastructural clues indicative of a plectin defect are a split just above the plasma membrane

of the basal cell with focal pseudojunctional appearance and hypoplasia of the inner plaque of

hemidesmosomes. An immunohistochemical indication of a plectin mutation may be absence

of plectin staining in the basal cell layer with monoclonal antibody 10F6.

94

Chapter 3

Acknowledgements

We are grateful to the patients and their families for their participation in this study. We would

like to thank Piet Toonder for the clinical photography, Janny Zuiderveen, Gonnie Meijer, and

Miranda Nijenhuis for their technical assistance and Jose Duipmans for her excellent help in

contacting the families. We thank Dr. K Owaribe, Nagoya, Japan (HD121, 1A8c, 1D1); Prof. E. B.

Lane, Dundee, UK (LL001, BL18); and Dr. A. Sonnenberg, Amsterdam, the Netherlands (58XB4,

clone 7) for their kind providence of antibodies. This study was supported by the J.P. Nater

Foundation (MCB) and DEBRA UK (WHIMcL, FJDS).

References

1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuderman L, Heagerty A, et al. The

classification of inherited epidermolysis bullosa (EB): Report of the Third International

Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol

2008;58(6):931-50.

2. Lane EB, Rugg EL, Navsaria H, Leigh IM, Heagerty AH, Ishida-Yamamoto A, et al. A

mutation in the conserved helix termination peptide of keratin 5 in hereditary skin

blistering. Nature 1992;356(6366):244-6.

3. Coulombe PA, Hutton ME, Letai A, Hebert A, Paller AS, Fuchs E. Point mutations in human

keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional

analyses. Cell 1991;66(6):1301-11.

4. Bonifas JM, Rothman AL, Epstein EH, Jr. Epidermolysis bullosa simplex: evidence in two

families for keratin gene abnormalities. Science 1991;254(5035):1202-5.

5. Rugg EL, Horn HM, Smith FJ, Wilson NJ, Hill AJ, Magee GJ, et al. Epidermolysis bullosa

simplex in Scotland caused by a spectrum of keratin mutations. J Invest Dermatol

2007;127(3):574-80.

6. Huber M, Floeth M, Borradori L, Schacke H, Rugg EL, Lane EB, et al. Deletion of the

cytoplasmatic domain of BP180/collagen XVII causes a phenotype with predominant

features of epidermolysis bullosa simplex. J Invest Dermatol 2002;118(1):185-92.

7. Pasmooij AM, van der Steege G, Pas HH, Smitt JH, Nijenhuis AM, Zuiderveen J, et al.

Features of epidermolysis bullosa simplex due to mutations in the ectodomain of type

XVII collagen. Br J Dermatol 2004;151(3):669-74.

8. Jonkman MF, Pas HH, Nijenhuis M, Kloosterhuis G, Steege G. Deletion of a cytoplasmic

domain of integrin beta4 causes epidermolysis bullosa simplex. J Invest Dermatol

2002;119(6):1275-81.

9. Gedde-Dahl T, Jr. Epidermolysis Bullosa. A clinical, genetic and epidemiological study. .

Baltimore and London: The Johns Hopkins Press 1971.

95

Plectin mutations in basal EBS

10. Koss-Harnes D, Jahnsen FL, Wiche G, Soyland E, Brandtzaeg P, Gedde-Dahl T, Jr. Plectin

abnormality in epidermolysis bullosa simplex Ogna: non-responsiveness of basal

keratinocytes to some anti-rat plectin antibodies. Exp Dermatol 1997;6(1):41-8.

11. Koss-Harnes D, Hoyheim B, Anton-Lamprecht I, Gjesti A, Jorgensen RS, Jahnsen FL, et al.

A site-specific plectin mutation causes dominant epidermolysis bullosa simplex Ogna:

two identical de novo mutations. J Invest Dermatol 2002;118(1):87-93.

12. 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(13):2461-72.

13. Elliott CE, Becker B, Oehler S, Castanon MJ, Hauptmann R, Wiche G. Plectin transcript

diversity: identification and tissue distribution of variants with distinct first coding exons

and rodless isoforms. Genomics 1997;42(1):115-25.

14. Koster J, van Wilpe S, Kuikman I, Litjens SH, Sonnenberg A. Role of binding of plectin

to the integrin beta4 subunit in the assembly of hemidesmosomes. Mol Biol Cell

2004;15(3):1211-23.

15. 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):387-99.

16. Eger A, Stockinger A, Wiche G, Foisner R. Polarisation-dependent association of plectin

with desmoplakin and the lateral submembrane skeleton in MDCK cells. J Cell Sci

1997;110 ( Pt 11):1307-16.

17. Andra K, Lassmann H, Bittner R, Shorny S, Fassler R, Propst F, et al. Targeted inactivation of

plectin reveals essential function in maintaining the integrity of skin, muscle, and heart

cytoarchitecture. Genes Dev 1997;11(23):3143-56.

18. 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(14):1724-35.

19. Pfendner E, Uitto J. Plectin gene mutations can cause epidermolysis bullosa with pyloric

atresia. J Invest Dermatol 2005;124(1):111-5.

20. Charlesworth A, Gagnoux-Palacios L, Bonduelle M, Ortonne JP, De Raeve L, Meneguzzi

G. Identification of a lethal form of epidermolysis bullosa simplex associated with a

homozygous genetic mutation in plectin. J Invest Dermatol 2003;121(6):1344-8.

21. Schuilenga-Hut PH, Vlies P, Jonkman MF, Waanders E, Buys CH, Scheffer H. Mutation

analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa

simplex and identification of novel mutations. Hum Mutat 2003;21(4):447.

22. Stephens K, Ehrlich P, Weaver M, Le R, Spencer A, Sybert VP. Primers for exon-specific

amplification of the KRT5 gene: identification of novel and recurrent mutations in

epidermolysis bullosa simplex patients. J Invest Dermatol 1997;108(3):349-53.

96

Chapter 3

23. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from

human nucleated cells. Nucleic Acids Res 1988;16(3):1215.

24. Sherry ST, Ward MH, Kolodov M, Baker J, Phan L, Smigielski EM, et al. dbSNP: the NCBI

database of genetic variation. Nucleic Acid Res 2001;29(1):308-11.

25. Jonkman MF, de Jong MC, Heeres K, Sonnenberg A. Expression of integrin alpha 6 beta 4

in junctional epidermolysis bullosa. J Invest Dermatol 1992;99(4):489-96.

26. Gache Y, Chavanas S, Lacour JP, Wiche G, Owaribe K, Meneguzzi G, et al. Defective

expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy. J

Clin Invest 1996;97(10):2289-98.

27. Wiche G, Becker B, Luber K, Weitzer G, Castanon MJ, Hauptmann R, et al. Cloning and

sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain

structure based on a central alpha-helical coiled coil. J Cell Biol 1991;114(1):83-99.

28. Green KJ, Virata ML, Elgart GW, Stanley JR, Parry DA. Comparative structural analysis of

desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family

involved in organization of intermediate filaments. Int J Biol Macromol 1992;14(3):145-

53.

29. Wiche G. Role of plectin in cytoskeleton organization and dynamics. J Cell Sci 1998;111 (

Pt 17):2477-86.

30. Foisner R, Feldman B, Sander L, Seifert G, Artlieb U, Wiche G. A panel of monoclonal

antibodies to rat plectin: distinction by epitope mapping and immunoreactivity with

different tissues and cell lines. Acta Histochem 1994;96(4):421-38.

31. Sonnenberg A, Liem RK. Plakins in development and disease. Exp Cell Res

2007;313(10):2189-203.

32. Rezniczek GA, Konieczny P, Nikolic B, Reipert S, Schneller D, Abrahamsberg C, et al. Plectin

1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fibers through multiple

interactions with beta-dystroglycan. J Cell Biol 2007;176(7):965-77.

33. Andra K, Nikolic B, Stocher M, Drenckhahn D, Wiche G. Not just scaffolding: plectin

regulates actin dynamics in cultured cells. Genes Dev 1998;12(21):3442-51.

34. Geerts D, Fontao L, Nievers MG, Schaapveld RQ, Purkis PE, Wheeler GN, et al. Binding of

integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not

interfere with intermediate filament binding. J Cell Biol 1999;147(2):417-34.

35. Wilhelmsen K, Litjens SH, Kuikman I, Tshimbalanga N, Janssen H, van den Bout I, et al.

Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal

linker protein plectin. J Cell Biol 2005;171(5):799-810.

36. Hijikata T, Nakamura A, Isokawa K, Imamura M, Yuasa K, Ishikawa R, et al. Plectin 1 links

intermediate filaments to costameric sarcolemma through {beta}-synemin, {alpha}-

dystrobrevin and actin. J Cell Sci 2008;121(Pt 12):2062-74.

97

Plectin mutations in basal EBS

37. Nikolic B, Mac Nulty E, Mir B, Wiche G. Basic amino acid residue cluster within nuclear

targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

J Cell Biol 1996;134(6):1455-67.

38. Steinbock FA, Nikolic B, Coulombe PA, Fuchs E, Traub P, Wiche G. Dose-dependent

linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments

through plectin’s intermediate filament-binding domain. J Cell Sci 2000;113 ( Pt 3):483-

91.

39. Janda L, Damborsky J, Rezniczek GA, Wiche G. Plectin repeats and modules: strategic

cysteines and their presumed impact on cytolinker functions. Bioessays 2001;23(11):1064-

9.

40. Osmanagic-Myers S, Wiche G. Plectin-RACK1 (receptor for activated C kinase 1)

scaffolding: a novel mechanism to regulate protein kinase C activity. J Biol Chem

2004;279(18):18701-10.

41. Osmanagic-Myers S, Gregor M, Walko G, Burgstaller G, Reipert S, Wiche G. Plectin-

controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress

response and migration. J Cell Biol 2006;174(4):557-68.

42. Rezniczek GA, de Pereda JM, Reipert S, Wiche G. Linking integrin alpha6beta4-based

cell adhesion to the intermediate filament cytoskeleton: direct interaction between the

beta4 subunit and plectin at multiple molecular sites. J Cell Biol 1998;141(1):209-25.

Figure S2. Conservation of plectin residues p.Arg2000, p.Thr2890 and p.Arg3527 involved in the PLEC1 missense mutations found in the EBS patients in this study.

2000EQA E L EAARQRQLAA E E EEQA E L EAARQRQLAA E E EEQA E L EA TRQRQLAA E E EAKA E E EA EK FRK LA L E E EVKA EQ EA EKQRQLA L E E E

3527YQRG Y F S E EMNR V LAD P SYQRG Y FD E EMNR V LAD P SYQRG Y FD E EMNR V LAD P S

2890F FD PN TH EN L T Y LQL L ERF FD PN TH EN L T Y LQL L ERF FD PN TH EN L T Y LQL L ERF I D PN TKD S L T Y S E L LDQ

Human_plectinMouse_plectinDog_plectinZebrafish_plectin homologueFrog_plectin homologue

Human_plectinMouse_plectinDog_plectinZebrafish_plectin homologue

Human_plectinMouse_plectinDog_plectin