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From the Department of Dermatology,*
Children’s
Hospital
Salzburg, Austria; the Institute of
Pathology,
General Hospital Salzburg,
Salzburg, Austria; the Department of Dermatology and Cutaneous
Biology,§
Thomas Jefferson University,
Philadelphia, Pennsylvania; and the Institute for Biochemistry and
Molecular Cell Biology,
Vienna
Biocenter, University of Vienna, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EB is a term used for a heterogeneous group of mechanobullous disorders, in which minor trauma leads to blister formation on skin and mucous membranes. Three major groups of EB have been defined according to the plane of split formation within the skin or mucous membranes: intra-epidermal cleavage in EB simplex, cleavage in the lamina lucida in junctional EB, and below the lamina densa in dystrophic EB.14 In a total of 10 different genes mutations have been found.15 EB simplex with late onset muscular dystrophy (EBS-MD), an autosomal recessive disease, is caused by nonsense mutations, out-of-frame deletions, and insertions in the plectin gene in most of the published cases.16 In one case, an in-frame deletion of three amino acids has been reported17 whereas there are no reports on in-frame insertions. In case of nonsense and out-of-frame mutations the functional consequences of mutations are thought to be mediated by nonsense-mediated mRNA decay, in case of in-frame mutations the pathophysiological consequences are less clear.
In this report we describe compound heterozygosity for a nonsense mutation and a 3-bp in-frame insertion in the plectin gene defining the smallest insertion mutation leading to a pathological phenotype in humans. This mutation leads to hemidesmosomal insufficiency in keratinocytes with blister formation and structural changes in skeletal muscle and nerves. Despite unaltered levels of plectin mRNA expression the amount of plectin protein is markedly reduced in skin and muscle. Protein overlay assays demonstrated increased self-aggregation of the mutated plectin molecules, thus providing a mechanistic explanation for the consequences of the insertion mutation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nuclear family consisted of the index patient (II-1), his
affected brother, his unaffected sister, and their parents, who are of
Caucasian origin. At the time of analysis patient II-1 was a 4-year-old
boy. Keratinocyte and fibroblast cultures were initiated from biopsies
of patient’s skin by sequential dispase and trypsin treatment and
culturing in keratinocyte growth medium and fibroblast growth
medium, respectively (BioWhittaker, Vervier, Belgium).
Muscle biopsies were obtained from left quadriceps muscle of the
patient II-1 (for patient numbering see Figure 4
) after gaining consent
by the parents. Anti-rat plectin antibodies 6C6 and 5B3 that
cross-react with human tissues were described elsewhere.18
Other antibodies were from the following sources and were used with the
following dilutions: anti-keratin 14 (Sigma, St. Louis, MO) dilution
1:100 in PBS; anti-keratin 5 (antibody AE14, kindly provided by H. Sun,
New York, NY) dilution 1:100 in phosphate-buffered saline (PBS);
anti-BPAG1 (antibody 233, kindly provided by J. Stanley, Philadelphia,
PA), anti-integrin {beta}4 (Chemicon, Temecula, CA) dilution 1:20 in PBS,
anti-BPAG2 (antibody HD 18, kindly provided by G. Giudice, Milwaukee,
WI) was used undiluted, anti-laminin 5 (GB3, Sera-Lab; Vienna, Austria)
dilution 1:20 in PBS, anti-type VII collagen (Chemicon) dilution 1:20
in PBS. Fluorescein-conjugated secondary antibodies were from Amersham
Pharmacia Biotech (Little Chalfont, UK) and were used 1:40.
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Immunofluorescence microscopy was performed on 5-µm cryostat sections of skin from two control patients, nonlesional skin as well as musculus vastus lateralis of patient II-1. Specimens were incubated with first-step antibodies for 1 hour at room temperature. 5B3 was diluted 1:2 in PBS. After three washes with PBS the respective fluorescein isothiocyanate-conjugated second step antibodies (1:100 in PBS) were added for 30 minutes at room temperature. Then, the sections were washed again, mounted, and evaluated under an immunofluorescence microscope (Zeiss, Vienna, Austria).
Immunohistochemistry and Myofibrillar Typing of Muscular Tissue
Tissue specimens were snap-frozen in liquid nitrogen and stored at -70°C. Sections (7-µm) were cut from each block and mounted. Protein expression was visualized with a three-step immunoperoxidase technique [streptavidin-biotin-peroxidase complex (S-ABC)]. Endogenous peroxidase activity was blocked by H2O2 methanol (0.3%) for 15 minutes. Sections were then rehydrated and rinsed with PBS (pH 7.2) throughout a 10-minute period. Nonspecific-binding sites were blocked with normal sheep serum (1:20 in PBS; DAKO, Glostrup, Denmark). All primary antibodies were incubated overnight at 4°C. The following primary antibodies were used: vimentin (dilution 1:200; DAKO); dystrophin 1-3 (dilution 1:4; Novocastra, Newcastle on Tyne, UK). The biotinylated second antibody (1:200, anti-mouse Ig from sheep; Amersham) was added and incubated for 30 minutes at room temperature. The S-ABC complex was visualized with aminoethylcarbazole in the presence of hydrogen peroxide. The reaction was stopped by immersion in PBS and finally sections were slightly counterstained with hematoxylin and mounted under coverslips with Kaiser’s glycerin-gelatine (Merck, Darmstadt, Germany).
To type muscular fibers a myofibrillar ATPase reaction at pH 4.2 and 9.4 was used according to Hayashi and Freimann.19
Western Blot Analysis
Confluent cells were washed with PBS and scraped off the plate. Cell pellets were lysed in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% (v/v) Triton-X 100, 0.1% (w/v) sodium dodecyl sulfate, 0.5 mmol/L ethylenediaminetetraacetic acid, 10 µmol/L leupeptin, 100 µmol/L phenylmethyl sulfonyl fluoride, 100 µmol/L dithiothreitol. Twenty µg of protein of control and patient II-1 keratinocytes were loaded on a 5% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose (Hybond C pure; Amersham Pharmacia Biotech) in 48 mmol/L Tris-HCl, 39 mmol/L glycine, 20% (v/v) MeOH, 0.037% (w/v) sodium dodecyl sulfate. The primary monoclonal antibody 5B3 was diluted 1:3 in blocking buffer (200 mmol/L Tris-HCl, pH 7.6, 137 mmol/L NaCl, 0.2% (w/v) I-Block, 0.1% (v/v) Tween 20). Immunodetection was monitored with the Western-Star chemiluminescent detection system (Tropix Inc., Bedford, MA) following the manufacturer’s instructions.
Electron Microscopy
Specimens were prepared for conventional transmission electron
microscopy using standard protocols. In brief, skin punch biopsies from
blister sites were immediately placed in the prefixation solution and
dissected in an oriented manner. Muscular tissue was also immersed in
prefixative solution and dissected at room temperature. The prefixation
solution consisted of a mixture of buffered formaldehyde: 0.5% (v/v)
and electron microscopic-grade glutaraldehyde: 1.5% (v/v), in 0.1
mol/L phosphate buffer (pH 7.5). After trimming, all specimens were
processed manually as follows: fixation in buffered glutaraldehyde (4%
of 0.1 mol/L phosphate buffer, pH 7.5, at room temperature for 5 to 6
hours). Rinsing in phosphate buffer (0.13 mol/L, pH 7.5, 4 x 5,
30, 5, and 5 minutes; 30-minute wash containing 50 mmol/L
NH4Cl), postfixation in buffered 1% osmium
tetroxide (0.13 mol/L phosphate buffer according to
Millonig20
room temperature for 1.5 hours); rinsing twice
again in phosphate buffer (0.1 mol/L, 5 minutes each); dehydration in
50% ethanol (EtOH), followed by 1% para-phenylenediamine in 70%
EtOH, rinsing specimens afterward in 70% EtOH; dehydration in a graded
series of EtOH; as an intermedium acetonitrile (3x pure, minimally 15
minutes each) was used. Infiltration and subsequent embedding in the
epoxy resin EPON 812 substitute (based on Glycidether 100; Serva,
Germany); polymerization at 37°C, 45°C, and 65°C for 
24 hours
each. Semithin sections (1-µm thick for localization purposes) were
stained with a modified methylene azure II basic fuchsin
sequence21
followed by ultrathin sectioning: 60 to 80 nm,
mounting sections on 75-mesh Formvar-coated copper grids, conventional
staining of ultrathin sections by a modified sequence, using a hydrous
solution of 0.05% tannic acid (6 to 8 minutes at room temperature),
1% (w/v) methanolic uranylacetate containing 50 µl/100 ml
(end-volume) of concentrated acetic acid (15 minutes at room
temperature), followed by lead citrate22
(2 to 3 minutes
at room temperature).
Genomic Analysis
Genomic DNA from leukocytes from peripheral blood of the probands was purified using a column method according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Primers for amplification of exon 9 (according to the sequence published by Smith and colleagues4 and McLean and colleagues,2 GenBank accession numbers U53204, U53834, and U63610, this exon is exon 10, the maternal mutation located there is 1008ins3. The paternal mutation is termed Q1408X at the start of exon 32) (GenBank accession number z543671) were 5'-GGCAGACCAACCTGGAGAAC-3' (nucleotides 1046 to 1065; z54367) and 5'-GTGTCGCATCCACTGAAGCA-3' (nucleotides 1302 to 1283; z54367). Primers for amplification of the mutated region in exon 31: 5'-TGAGTGAACTGTGCCGGTGC-3' (nucleotides 11,814 to 11,833; U63610) and 5'-TTTGTGCCTCAGCCTCCTCC-3' (nucleotides 4876 to 4857; z54367). Polymerase chain reactions (PCRs) were performed at 95°C denaturing temperature, 60°C and 62°C annealing temperature, respectively, and 68°C extension temperature using Expand Long-Range PCR kit (Roche Molecular Biomedicals, Indianapolis, IN). Amplified DNA was analyzed by conformation-sensitive gel electrophoresis following a standard protocol.23 Heteroduplex bands were automatically cycle-sequenced with a ABI Prism 377 (ABI, Foster City, CA) using the above-mentioned 5' primers to detect mutations in exon 9 and exon 31, respectively. Primers and PCR conditions for sequencing were the same as used for initial PCR reactions. Restriction enzyme digests were performed according to the manufacturer’s protocols (New England Biolabs, Beverly, MA). The DNA fragments were analyzed on a 4% agarose gel.
RNA Isolation and RNase Protection Assay
Total RNA was isolated from cultured fibroblasts of patient II-1 by the method of Chomczynski and Sacchi.24 RNase protection analysis was performed as previously described25 with the exception that hybridization was done at 60°C. Ten µg of total RNA was used per lane. Probes specific for exon E1 (human plectin cDNA nucleotides 34 to 141; z54367) and E32 (nucleotides 13,650 to 13,906; z54367) were subcloned from genomic DNA clone pCGL66 (containing nucleotides 1 to 141 of exon E1 and 600 nucleotides of upstream sequences) and cDNA clone pCGL53,1 respectively. The probe specific for plectin’s alternative first exon E1c (human plectin cDNA, nucleotides 73 to 220; NM000445) was subcloned from cDNA clone pIK7 (containing E1c-E3 of human plectin cDNA). The DNA fragments were inserted into the polylinker of the plasmids pSP64 or pSP65 (Promega, Madison, WI) in opposite orientation to the direction of SP6 transcription. After linearization at a suitable restriction site downstream of the plectin insert in vitro transcription with SP6 polymerase was performed to produce an antisense riboprobe. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe was shortened to produce a signal short enough to serve as loading control. Quantification of transcript levels was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), the values were normalized against the protected GAPDH fragment and corrected for the G content of the different riboprobes. A radioactively labeled HpaII digested pUC18 DNA was run simultaneously to estimate the size of the protected fragments.
Cloning and Expression of Recombinant Proteins
A mouse plectin cDNA construct comprising exons 2 to 15 (GenBank
accession number z54367) flanked by in-frame EcoRI
restriction sites was generated by PCR using mouse cDNA not containing
any of the alternative 
-exons13
as template and primers
mEx2fw (5'-cgg gaa ttc GAT GAA CGA GAC CGT GTG CAG-3', corresponding to
nucleotides 523 to 543 in the human sequence; z54367) and mEx15rev
(5'-aag gaa ttc ACC CCG GGT GGC AGG GGA G-3', nucleotides 2157 to 2175;
z54367). To introduce the additional leucine into the sequence, we took
advantage of the immediate proximity of a BamHI restriction
site to the insertion point and a unique BglII restriction
site within exon 6. PCR mutagenesis using primers mEx6fw (5'-cgg gaa
ttc CAG ATC TCA GAC ATT CAG-3', nucleotides 844 to 861; z54367) and
mEx9leu ins (5'-ccg gaa ttc CCG GAT CCA TTG Cag
cAG CAG CAG CAA CAC AAG CTC CCG-3', nucleotides 1264 to 1295; z54367)
was performed. The amplified mutagenized PCR product was subcloned,
sequenced, and the BglII/BamHI restriction
fragment from the exon 2 to 9 wild-type construct replaced with the
mutated fragment. Constructs were then extended to the beginning of
exon 15 and subcloned into the unique EcoRI sites of the
HIS-tag expression vector8
pBN120. Proteins (2 to 15 wild
type and 2 to 15 mutated) were expressed in E. coli
BL21(DE3) and affinity purified under nondenaturing conditions. The
HIS-tagged cytoplasmic domain of the integrin {beta}4 subunit protein
({beta}4cyt), starting with the first pair of fibronectin type III
repeats, was described earlier.9
Rabbit skeletal muscle
actin was from Sigma Chemical Co. (St. Louis, MO).
In Vitro Protein Interaction Assay
Influences of the mutation on the properties of plectin were assessed by a microtiter-plate format protein-protein interaction assay that had been successfully used to characterize interactions of N-terminal plectin fragments with the integrin subunit {beta}49 and actin.13 Expressed proteins were labeled with Eu3+-labeling reagent (Wallac, Turku, Finland) as previously described.9 Microtiter plates were coated with 100 µl of 100 nmol/L protein in 25 mmol/L sodium borate buffer, pH 9.2, overnight at 4°C. Blocking was performed with 4% (w/v) bovine serum albumin (BSA) in overlay buffer [50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol, and 0.1% (v/v) Tween 20] for 1 hour, followed by incubation with different concentrations of Eu3+-labeled proteins (in 100 µl of overlay buffer) for 90 minutes at room temperature. After extensive washing with overlay buffer, the amount of bound proteins was determined by measuring Eu3+-fluorescence with a Delfia time-resolved fluorometer (Wallac). The fluorescence values were converted to concentrations by comparison with an Eu3+ standard.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Patients II-1
and II-2 showed blistering since birth. Blisters tended to form
erosions but were also present as blood blebs on fingers and toes
(Figure 1, A and B)
. The blisters healed
without scarring. In addition, nails were dystrophic, but mucous
membranes, teeth, and hair were not affected. Clinical tests including
neuropediatric evaluation, evaluation of muscle strength, and cranial
CT did not reveal any abnormalities.
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. Control antibodies including
antibodies detecting BPAG1, BPAG2, integrin {beta}4, laminin 5, and type
VII collagen did not reveal significant alterations.
Immunohistochemical analysis of skeletal muscle revealed normal tissue
distribution of ATPase, vimentin, and dystrophin in a biopsy of
musculus vastus lateralis from patient II-1, whereas plectin mAb 5B3
binding was absent. In addition, an irregular distribution of nuclei,
variation in size and shape, was seen in the muscle fibers as compared
to a control muscle. Furthermore, in indirect immunofluorescence
microscopy analysis of patient muscle mAb 5B3 staining was absent.
Control muscle cells showed typical predominant staining of Z-lines in
longitudinal sections and peripheral and sarcoplasmic staining in cross
sections (data not shown). Western blot analysis of cultured patient
keratinocytes with 5B3 gave only a very faint band (Figure 2b)
. In
addition, several bands of 40 to 50 kd suggestive for degradation
products were seen using antibody 6C6 recognizing an epitope in
plectin’s rod domain upstream of that of 5B3 (data not shown).
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. Keratin
filaments were not inserting in the residual hemidesmosomes with a few
exceptions. Also, desmosomes in the basal layer and in the higher
layers of the epidermis were hypoplastic (not shown). Sections of
skeletal muscle showed moderate pathological changes: Z- and I-band
alterations and disoriented muscle fibers were seen (Figure 3b)
.
Mitochondria were often misplaced and degenerating (Figure 3, c and d)
.
Around muscle fibers muscular nerves showed axons with adaxonal
microlamellar inclusions and myelin-like lamellar bodies (Figure 3, e–g)
. The axonal ending exhibited discrete signs of degeneration such
as the basal lamina redundant foldings (not shown).
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. The insertion of GCT is placed after
four other GCTs and might have been caused by slipped mispairing. It
leads to the insertion of another leucine in a row of four leucines.
Another heteroduplex formation was observed at the beginning of exon
31. Here a C-to-T transition leads to a stop codon designated Q1518X.
The paternal mutation Q1518X was verified by digestion of the
respective exon 31 fragment with BfaI (Figure 4C
-I). Because
of the creation of a new BfaI restriction site by the
mutation this digest leads to the heterozygous occurrence of two
fragments of 325- and 185-bp length in the patients II-1, II-2, the
unaffected sister II-3, and the father. These fragments were not
observed in the mother and in 80 control alleles. The maternal mutation
1287ins3 was verified by PvuII digestion of a PCR fragment
spanning exon 9 (Figure 4C
-II). This digest leads to two fragments of
361 bp and 57 bp in length in the father, the unaffected sister and 80
control alleles. In the mother, patient II-1 and patient II-2 a
heterozygous 60-bp fragment created by the 3-bp insertion was seen in
addition. To determine the mRNA levels in fibroblasts from patient II-1
and unaffected controls RNase protection assays were performed using
riboprobes specific for the alternative first N-terminal exons E1 and
E1c and the C-terminal exon E32. This analysis showed similar levels of
plectin E1c transcripts in patient and control cells (Figure 4D
, left).
Furthermore, the ratio between the levels of alternative plectin
transcripts E1 and E1c was similar to that found in control fibroblasts
(not shown). Also, no apparent reduction in the levels of exon 1- and
exon 32-specific transcripts was noticed suggesting that in patients’
fibroblasts full-length mRNA was produced (Figure 4D
, right).
The insertion mutation of the N-terminal globular domain lies in close
proximity to the multifunctional actin binding domain, encoded by exons
2 to 8. Therefore, it was of interest to analyze whether this mutation
affected the binding of plectin to the established binding partners in
this region, actin13
and integrin {beta}4.9,10
Wild-type and mutated plectin proteins encoded by exons 2 to 15 were
generated by PCR-mediated mutagenesis and expressed as N-terminal
HIS-tag fusions in Escherichia coli. During the isolation
and purification of the mutated recombinant protein from bacteria we
found it to be much less soluble than its wild-type counterpart, with
expression levels being equal. Wild-type and mutated proteins were
coated (BSA served as the negative control) and overlaid with
Eu3+-labeled recombinant integrin {beta}4 or
skeletal muscle actin. Although no significant changes in binding to
skeletal muscle actin could be observed (data not shown), binding of
the mutated protein to integrin {beta}4 was 30% reduced when compared
to binding to the wild-type protein (Figure 5A)
. However, when integrin {beta}4 was
coated and overlaid with Eu3+-labeled recombinant
plectin proteins, we found the mutated protein to bind significantly
stronger (data not shown). Because the latter finding could be an
artifact of solubility of the mutated plectin protein we speculated
that the mutated protein had a potential for self-binding/aggregation.
To test this possibility, we performed assays in which wild-type or
mutated proteins were coated and overlaid with
Eu3+-labeled versions of the same kind. As shown
in Figure 5B
, no self-binding activity was observed for the wild-type
protein, whereas the mutated protein exhibited strong self-binding
activity. Both proteins gave the same low background levels when BSA
was coated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In-frame insertion mutations rarely cause human pathology. The combination of a stop codon and a three amino acid insertion in the insulin receptor gene led to severe insulin resistance.27 On the other hand, the insertion of three and six amino acids in the pro-opiomelanocortin gene does not cause a pathological phenotype.28 To date, we are not aware of other in-frame insertion mutations in EB. Together with a 3-bp insertion in the human phythanoyl-CoA hydroxylase gene causing Refsum’s disease29 the mutation 1287ins3 defines the smallest possible amino acid insertion causing a pathological phenotype.
The pathophysiological consequences of in-frame mutations are probably mediated by disruption of functional domains. However, to date no experimental evidence for this assumption has been published to our knowledge. The N-terminal globular domain of plectin harbors an actin binding domain12 and regions for integrin {beta}4 binding.9,10 The importance of this region has also been demonstrated by a three amino-acid deletion in the N-terminal globular domain of plectin in a patient with EBS with muscular dystrophy.17 In that study the functional effects of the mutation were not analyzed. Using a protein overlay assay we have observed increased self-association of the recombinant plectin mutant protein mimicking the insertional mutation of the patient. A likely explanation for this observation is that in the mutated protein the insertion of an additional leucine into a stretch of four existing consecutive leucines leads to an increase in hydrophobicity of this region resulting in conformational changes in the molecule. This could lead to reduced integrin {beta}4-binding and a possible surface exposure of hydrophobic amino acids normally buried within the naturally folded proteins, which in turn could be responsible for the observed self-binding and decreased solubility. We speculate that these self-aggregated plectin molecules are degraded rapidly, explaining the greatly reduced amount of plectin protein in our patient cells. To our knowledge, these results demonstrate the functional consequences of an in-frame insertion mutation for the first time and suggest that the protein overlay assay is a valuable tool in defining the functional deficiencies caused by in-frame mutations.
In summary, we describe here that a combination of a stop codon in one allele and a single amino acid insertion in the other allele of the plectin gene can lead to loss of plectin protein possibly caused by increased self-aggregation and subsequent degradation. This leads to blister formation in the skin and ultrastructural pathology in muscles and nerves. A follow-up study on these patients including re-evaluation of muscle tissue when clinically overt muscular weakness starts might further help to reveal the pathophysiology of muscular disease in EB.
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Acknowledgements |
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Footnotes |
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Supported by the Medizinische Forschungsgesellschaft Salzburg (to A. K., A. H., and R. H.); a predoctoral fellowship from the Austrian Academy of Sciences (to G. A. R.); by grants from the Austrian Science Research Fund (to G. W.); the Austrian National Bank; and Verein zur Erforschung von Muskelkrankheiten bei Kindern.
Accepted for publication October 24, 2000.
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