university of groningen epidermolysis bullosa simplex ... · 59 krt5 and krt14 mutations in 75% of...

University of Groningen

Epidermolysis bullosa simplexBolling, Maria Caroline

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Mutations in KRT5 and KRT14 cause epidermolysis bullosa simplex

in 75% of the patients

MC Bolling1, HH Lemmink2, GHL Jansen2, MF Jonkman1

1 Center for blistering skin diseases, Department of Dermatology2 Department of Genetics, University Medical Center Groningen,

University of Groningen, the Netherlands

Submitted

58

Chapter 2

Abstract

Background Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis which

is inherited as a predominantly autosomal dominant trait caused by mutations in the genes

KRT5 and KRT14 encoding the basal epidermal keratins 5 (K5) and 14 (K14). Three main clinical

subtypes of EBS exist, differing in onset, distribution and severity of skin blistering. Previous

reports of KRT5 and KRT14 mutations suggest a correlation between the location of the mutation

and the severity of the associated EBS phenotype.

Objectives The prevalence of KRT5/14 mutations and the genotype-phenotype correlation in the

largest tissue confirmed EBS population is investigated.

Methods KRT5 and KRT14 genomic DNA and cDNA sequences of 65 clinically well defined

unrelated EBS probands were amplified and then subjected to direct sequencing and product

length analysis. Immunofluorescence microscopy on patients’ skin biopsies with antibodies

against K5 and K14 was performed to study protein expression.

Results In 49 of 65 (75%) probands 37 different KRT5 and KRT14 mutations were identified, of

which 11 were novel. Mutations affecting the highly conserved helix boundary motifs of the rod

domains of K5 and K14, and the K14 helix initiation motif in particular, were associated with the

severest, EBS Dowling Meara, phenotype. In 19 EBS probands (39%) the mutation was de novo.

In 16 probands (25%) KRT5 or KRT14 mutations were excluded.

Conclusions The phenotype-genotype correlation observed in this large EBS population

underscores the importance of helix boundary motifs for keratin assembly. The high percentage

of biopsy confirmed EBS probands without KRT5 or KRT14 mutations indicates genetic

heterogeneity in EBS. Alternative gene candidates are discussed.

59

KRT5 and KRT14 mutations in 75% of EBS patients

Introduction

Epidermolysis bullosa simplex (EBS) is the most common hereditary skin blistering disease

with an estimated prevalence of 1 in 25.000-50.000.1-3 EBS is characterized by cleavage through

basal epidermal keratinocytes upon minor trauma leading to skin blistering in a wide range of

severity (figure 1).4 EBS-localized (EBS-loc, former EBS Weber-Cockayne) is the mildest form with

onset of blistering in infancy mainly affecting hands and feet. EBS-generalized-non Dowling

Meara (EBS-gen-nDM or EBS-gen, former EBS Koebner) is characterized by generalized blistering

usually present from birth. EBS Dowling-Meara (EBS-DM) is the most severe subtype with severe

neonatal generalized circinary or herpetiform grouped blisters often affecting the mucosae

as well, with later on development of palmoplantar keratoderma. Other subtypes are EBS

mottled pigmentation (EBS-MP), EBS migratory circinate (EBS-migr) and autosomal recessive

EBS (EBA-AR). In most cases EBS is inherited as an autosomal dominant disorder and caused

by mutations in KRT5 and KRT14 encoding the basal epidermal keratins keratin 5 (K5, a type II

keratin) and keratin 14 (K14, a type I keratin).5-7 K5 and K14 form parallel coiled-coil heterodimers

by means of their α-helical rod domains and through higher order assemble into a dynamic

tonofilament cytoskeleton to provide strength and flexibility to the basal keratinocytes (for

review see 8-11). From all reported KRT5 and KRT14 mutations a genotype-phenotype correlation

between the location of the mutation and the severity of the phenotype can be deducted. Most

mutations affecting the highly conserved helix boundary motifs (HBMs) of the rod domains of

K5 and K14, that are important for proper filament assembly, are associated with the severe

EBS-DM phenotype while mutations outside these domains tend to be associated with milder

phenotypes (see Human Intermediate Filament Database, www.interfil.org).12

60

Chapter 2

Figure 1. Clinical subtypes of EBS. Upper row, starting left: EBS-loc with blistering confined to hands and feet, EBS-gen with generalized non-herpetiform blistering, and EBS-DM with circinary/herpetiform blistering on erythematous skin. Lower row, starting left: mottled pigmentation in body folds of a patient with EBS-MP and mutation KRT5:p.Pro25Leu, EBS-migr with migratory circinate and annular erythema with blistering on the border of the erythema, and electron microscopy of a skin sample of an EBS-DM patient with clumping of keratin.

Here we present the results of KRT5 and KRT14 mutation analysis in the largest group of EBS

patients reported thus far consisting of 65 unrelated EBS families. The clinical diagnosis of EBS

was confirmed in skin biopsies of the probands showing an intraepidermal plane of cleavage

through the basal keratinocyte layer. Thirty-seven different mutations were detected, of

which 11 are novel and 26 have been previously described. The effects of the detected KRT5

and KRT14 mutations on the clinical and molecular level, in conjunction with the previously

reported data on EBS, will be discussed. KRT5 or KRT14 mutations were identified in 75% of all

EBS families, which leaves 25% of EBS cases unsolved on the molecular level. Confirmation of

the clinical diagnosis of EBS by mutation detection provides certainty about the diagnosis of

EBS, and insight in the mode of inheritance and clinical prognosis. In addition, as gene therapy

comes closer to application in clinical practice identification of the gene involved in inherited

disease becomes essential. Therefore it is important to discover the other genetic causes of EBS.

Candidate genes will be discussed in this article.

Materials and methods

Patients

Between 1989 and 2009 a total of 65 families with EBS were identified at the Center of Blistering

Diseases, Department of Dermatology, Groningen, the Netherlands. Fifty-two EBS families were

of Dutch origin. Three families were originally from Belgium, one from Finland, one from India,

one from Korea and seven from Middle-East countries. The patients were all clinically assessed

loc gen DM

MP migr DM

61


by the same expert (M.F.J.). The diagnosis was based on clinical features, immunofluorescence

antigen mapping and electron microscopic (EM) analysis revealing a basal intraepidermal

split in lesional skin biopsy. For classification the latest diagnostic criteria were used for basal

EBS subtypes (and see also figure 1).4 A clinical diagnosis of EBS-DM was made if at least one

of the following features was present: 1. severe generalized herpetiform blistering usually

present from birth, and 2. tonofilament clumping in (supra-)basal cells observed by EM. EBS-

gen is characterized by generalized non-herpetiform blistering with onset at birth without

tonofilament clumping. EBS-loc was defined as blistering confined to hands and feet, usually

with seasonal variation being more severe during summer. EBS-MP is characterized by a mild

EBS-gen or an EBS-loc blistering phenotype in combination with reticulated hyperpigmentation

with preference for the body folds and extremities. Routinely 4-mm (for immunofluorescence)

and 2-mm (for EM) punch biopsies were taken from lesional or rubbed skin, and of healthy (non-

blister) skin. Immunofluorescence and EM were performed as previously described (Jonkman, et

al., 1999). For immunofluorescence antibodies BL18 (gift from Dr. P. Ogden, Dundee, UK) against

K5, LL001 (gift from Prof. B. Lane, Dundee, UK) against K14, GB3 (Abcam, Cambridge, UK) against

laminin-332, and LH7.2 (gift from Prof. I. Leigh, London, UK) against type VII collagen were used

to determine the level of blister formation and the keratin expression in basal cells. Informed

consent was provided by all patients, family members and healthy control participants. The

local medical ethical committee approved all conducted studies in this article. The study was

conducted according to the ethical principles provided by the Declaration of Helsinki.

Mutation detection

Genomic DNA (gDNA) was extracted from peripheral blood lymphocytes from patients and family

members using 6 M NaCl and chloroform.13 Polymerase chain reaction (PCR) amplification of

exons 1-9 of KRT5 (GenBank NM_000424.3) and exons 1-8 of KRT14 (GenBank NM_000526.3) was

performed according to protocols as previously described.14, 15 PCR products were subsequently

purified with the Qiagen Quick PCR Purification Kit (Qiagen) and directly sequenced using an

automated ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Novel KRT5

and KRT14 mutations were excluded in 100 alleles of control gDNA samples. Mutations are

numbered according to the above mentioned cDNA GenBank accession numbers with +1 as

the adenine nucleotide of the ATG startcodon. Conservation of the residues substituted in the

novel missense mutations was investigated by collecting K5 and K14 homologues with an NCBI-

BLAST search. Comparative/multiple sequence analysis was then performed by ClustalW2 and

visualized with a Java viewer (Jalview).

RNA analysis

To screen for large deletions or duplications in the KRT5 or KRT14 genes we analysed RNA

obtained from nonlesional fresh frozen skin samples of the probands by RT-PCR. Per proband

four cryosections of 10 µm thickness were cut and transferred with a sterile needle into

62

Chapter 2

lysis buffer (Stratagene Europe, Heidelberg, Germany). RNA was extracted and cDNA was

subsequently synthesized according to protocols provided by the manufacturer (Absolutely

RNA Microprep Kit protocol, Stratagene Europe and Invitrogen, Breda, The Netherlands). The

resulting cDNA was PCR amplified with forward primer sequences located in exon 1 and reverse

primer sequences located in the most 3’end of the KRT5 and KRT14 cDNA sequences. Primer

sequences and PCR amplification protocols are presented in supplemental table S1. The length

of the amplified cDNA fragments was analyzed on 1% agarose gels.

Supplemental Table S1. Primer sequences and conditions for PCR amplification of KRT5

and KRT14 cDNA sequences

Primers Primer sequence (5’- 3’) PCR conditions

KRT5c- F cDNA GTC TCG CCA GTC AAG TGT G 1x: 94ºC for 2’35x: 94ºC for 10”, 64ºC for 30”, 68ºC for 90”1x: 68 ºC for 10’

KRT5c-R cDNA TTT GAC GCT GGA GCT GCT ACC C

KRT14c-F cDNA AGC TCC ATG AGG GCT CCT G 1x: 94ºC for 2’35x: 94ºC for 10”, 64ºC for 30”, 68ºC for 90” 1x: 68ºC for 10’

KRT14c-R cDNA CCA TCG TGC ACA TCC ATG AC

63


Figure 2. Schematic representation of the basal keratins (keratin 5 in (a), keratin 14 in (b)) with in light green the non-helical head domains, the α-helical rod domains in light blue, and in pink the helix boundary motifs. The mutations detected in the present study are illustrated above the protein with the novel mutations in a box. Below the protein the previously reported mutations are shown (the sum of different mutations with their associated phenotypes per keratin domain). *Frame shift mutations.

Results

Mutations were identified in 49 out of 65 EBS families (75%). Thirty-seven different mutations

were found of which 22 mutations resided in KRT5 and 15 mutations in KRT14 (figure 2 and table

1 at the end of the chapter). A total of 11 novel mutations were found of which seven in the KRT5

and five in the KRT14 gene. These mutations were excluded in unaffected family members. In 23

of the 49 families in which a mutation was found the proband was the first affected individual in

the family (47%). In 19 of these sporadic probands DNA from both parents was available and the

mutation could be excluded, indicating de novo mutations (39%). Twenty-seven of 37 mutations

.

. .... loc, early onset

.1

3

H1 1B 2A H21A 2B

p.P25L(x3)

p.N177S

p.K404E

p.A438Dp.I467M

p.G476Np.E475K

p.V186_Q189del

a K5 loc

DM

gen-nDM

2*3*3

4

8 1

1

1

141

131* 7*

11*1*

1*

.

11*

MP (Mottled Pigmentation)

DDD (Dowling-Degos Disease)

MCE (Migratory Circinate Erythema)

2 42 3

c.1635delGp.N329Kp.R331H

p.A180D

p.T151P

p.L149Pp.K199M

p.R187P(x4)p.V323G

p.K443Np.E170G

c.1649delG

4

b K14 recessive gen-nDM

2A1B

p.R125C(x4)p.R125H

p.L130P

1A 2B

p.R388C(x3)

p.L419Q

c.927+1G>A

p.I377Np.I176VfsX2, I176PfsX30(x3) p.411delE

1*

1*

. NFJS=Naegeli-Franceschetti-Jadassohn Syndrome

. DPR=Dermatopathia Pigmentosa Reticularis

1

1

123

111

3 35* 7

5*1

214

p.L408M

1*

1

3 2 1*1*

p.A247_K250delinsEp.128delS p.I412K

c.927+1G>T

p.A180P

p.Q191P

2*

2* 1

1

2

p.T414AfsX25

64

Chapter 2

were heterozygous missense mutations (73%). One previously reported autosomal recessive

splice-site mutation caused recessive gen-nDM EBS due to loss of K14 expression (see also16, 17).

The mutations were excluded in 100 healthy control alleles.

The EBS-DM phenotype was clinically clearly discernable and confirmed by detection

of tonofilament clumping in tissue samples in all 11 probands using EM. The other patients

formed a spectrum in severity of disease varying from early-onset persistent generalized

blistering, designated EBS-gen, to the one end, and blistering confined to hands or feet

beginning at the age when the child started to walk, and with clear improvement during winter

months, designated EBS-loc, at the other end of the spectrum. In between are patients with mild

generalized blistering starting from birth, which markedly improved over the years, so that later

in life blistering was confined to hands and/or feet. This group of patients is separated as early-

onset EBS-loc. No correlation could be observed between the location of the mutation in K5/

K14 and EBS-loc versus early-onset EBS-loc. The characteristics of the identified KRT5 and KRT14

mutations and the clinical features of the EBS patients are presented in table 1. Figure 2 provides

a schematical illustration of the K5 and K14 proteins with the mutations identified in this study,

including the previously reported mutations. Conservation of the wild-type residues affected by

the novel missense mutations is illustrated in figure 3.

KRT5 mutations

Twenty-two KRT5 mutations were found in 27 families. Fifteen of these mutations have been

described before. In three mutations (p.Glu170Gly in EB190 with EBS-loc, p.Ala180Asp in EB201

with EBS-gen, and c.1635delG in EB218 with EBS-gen) the EBS phenotype diagnosed in this study

was different from the EBS subtype in patients with identical mutations described in previous

reports, respectively EBS-gen, EBS- DM, and EBS-loc.18-20 Two affected sisters in family EB116

carried mutation p.Ile467Met while genomic DNA of their unaffected parents was negative for

this mutation, suggesting germline mosaicism. This was the only family in which more than one

sibling was affected without the parents carrying the mutation in their genomic DNA.

Mutations p.Pro25Leu and c.1649delG in the non-helical head and tail domains of K5

were associated with the rare phenotypes EBS with mottled pigmentation (EBS-MP) and EBS

with migratory circinate erythema (EBS-migr), respectively, as described in earlier reports.21-24

The affected persons in family EB218 with mutation c.1635delG and an EBS-gen phenotype

additionally had pigmentation abnormalities with hypo-and hyperpigmented spots on legs and

trunk. Two de novo mutations in the 2B domain of K5 (p.Ala438Asp and p.Glu475Lys) and one

autosomal dominant mutation in the HIM of K5 (p.Ala180Pro) were found in patients with EBS-

DM. Of note, affected persons of family EB073 with EBS-DM carrying mutation KRT5:p.Ala438Asp

were previously mistakenly reported to have an EBS-loc phenotype.14

65


Novel KRT5 mutations

Mutation p.Leu149Pro was found in a single Belgian family (BE03) with autosomal dominant

early-onset EBS-loc. This mutation is located in the non-helical H1 domain preceding the

HIM of the K5 rod domain. Residue p.Leu149 is conserved among different species and other

intermediate filaments. Substitution by a proline residue means a considerable conformational

change of the protein.

The p.Thr151Pro mutation, which is also localized in the K5 H1 domain, was found

in all persons affected with EBS-loc in family EB135. The p.Thr151 residue is conserved among

mammalians. In this case a polar and hydrophilic threonine is substituted for a non-polar and

hydrophobic proline residue which causes a conformational change. This mutation was not

found in normal controls.

Two different substitution of the highly conserved alanine residue at position 180 of

K5 were found. Mutation p.Ala180Asp was de novo in a newborn with EBS, gen-nDM (EB201). In

a previous study this mutation was reported to be associated with a DM phenotype18. However,

EM analysis of affected skin samples of proband EB201 did not show keratin clumps. Of note,

this patient was a neonate and possibly EM analysis at an older age will reveal keratin clumping.

Mutation p.Ala180Pro in proband EB209 was associated with an EBS-DM phenotype as clumping

was observed on EM analysis in skin samples.

Patients of four unrelated families (EB094, EB038, EB136, and EB144) with mild EBS-loc

carried the novel p.Arg187Pro mutation. The p.Arg187 residue is part of the 1A domain of the

K5 rod and comprises a CpG dinucleotide. Residue p.Arg187 is well conserved and substitution

by proline leads to a change of polarity and hydrophobicity of the protein at this position.

Furthermore, substitutions by a proline are often pathogenic as they cause kinking in the

peptide chain and may disrupt α-helical and β-sheet structures of proteins.

Both mutations p.Lys199Met and p.Lys443N involve conserved lysine residues in

the rod domain of K5 (1A and 2B, respectively) and the substitution by a methionine and an

asparagine respectively leads to a change in polarity and pH. However, both residues 199 and

443 are not part of the HBMs and associated with EBS-loc phenotypes.

In addition to two previously described L12 domain mutations (p.Asn329Lys and

p.Arg331His) we also found a novel de novo missense mutation affecting the non-helical linker

L12 domain of K5, p.Val323Gly. Similar to other linker domain mutations, mutation p.Val323Gly

caused mild EBS-loc. Residue p.Val323 is well conserved, but the physical-chemical changes

caused by the substitution are predicted to be minor. The mutation was proven to be de novo

and excluded in normal controls.

KRT14 mutations

Fifteen different KRT14 mutations were found, of which four were novel, in patients from 22

families. The phenotypes of the patients carrying mutations reported before were similar to the

ones in the previous reports. Four different mutations (p.Arg125Cys, p.Arg125His, p.Ser128del,

66

Chapter 2

p.Leu130Pro) identified in seven probands with the severe EBS-DM phenotype were located

in the HIM of K14. One mutation in the K14 HTM (p.Leu419Gln) was associated with an EBS-

DM phenotype as well (family EB069). Another mutation affecting both the K14 HTM and tail

(c.1240-1249del10, p.Thr414AlafsX25) was associated with clinical features resembling EBS-DM,

but with the for EBS-DM pathognomonic keratin clumping in patient’s skin biopsies (patient

EB205).

Novel KRT14 mutations

An in frame deletion-insertion mutation (c.740_748del9ins3) was found in patients of family

EB108 showing a mild EBS-loc phenotype. This mutation results in deletion of three amino acids

with insertion of a glutamic acid in the 1B domain of the K14 protein.

In family EB096 the proband and her son with early-onset EBS-loc carried a

heterozygous donor splice site mutation (c.927+1G>T) in intron 4 which is predicted to cause

aberrant splicing. Interestingly, another nucleotide change at the same position was found de

novo in proband EB128 with EBS-loc (c.927+1G>A). This latter mutation was previously described

in a family with EBS-loc and RNA analysis had revealed an inframe nine-bp skip in the transcript

by use of an alternative donor splice site25.

Mutation p.Ile412Phe was found in proband EB069 and his daughter with EBS-loc.

This mutation involves a highly conserved isoleucine residue at the a position of the heptad

structure of the α-helical structure of the 2B domain of K14. The physical-chemical characteristics

of isoleucine and phenylalanine are quite similar: both neutral, non-polar and hydrophobic.

Mutation c.1240-1249del10 was found de novo in a baby (EB205) with a clinical

phenotype of EBS-DM with fluctuating widespread blistering in a grouped pattern on an

erythematous underground rendering it an inflammatory aspect. However, no keratin clumping

was observed in a patient’s skin sample and therefore the phenotype was defined as EBS-gen.

The mutation causes a frameshift and a subsequent PTC after 25 amino acids (p.Thr414AlafsX25).

This mutation is predicted to cause an aberrant amino acid sequence in the HTM (2B) of K14 and

truncation of a large part of the K14 tail.

67


Figure 3. Conservation of the K5 and K14 residues that are affected by the novel missense mutations in this study. Above the amino acid sequences the residue numbers are depicted. Colours are according to the ClustalX mode of Jalview. GenBank: human K5, NP_000415.2; human K3, NP_476429.2; human K1, 006112.3; human K14, NP_000517.2; human K15, NP_002266.2; human K10, NP_000412.3; human desmin: NP_001918.3, human vimentin: NP_003371.2; chimpanzee K5, BAF62401; bovine K5, NP_001008663.1; mouse K5, NP_081287.1; rat K5, NP_899162.1; fish K5, NP_571231.1; frog K5, NP_001072377.1; chimpanzee K14 predicted, XP_511488.2; mouse K14, NP_048654.1; rat K14, NP_001008751.1; chicken K14, NP_001001311.2.

No KRT5 or KRT14 mutations

KRT5 or KRT14 mutations were excluded in 16 EBS probands (25% of the total sum of EBS families).

EBS was confirmed by cleavage through the basal cell layer in skin biopsy. Immunofluorescence

staining of skin biopsies showed presence of K5 and K14 proteins and additional analysis of

the complete KRT5 and KRT14 transcripts did not indicate heterozygous inframe deletions or

insertions as no additional bands were observed by analyzing the PCR fragments of RT-PCR on

agarose gel (data not shown). The phenotypes of these 16 non-KRT5/KRT14-mutation-EBS cases

varied from EBS-DM (n=2) to EBS-loc (n=12). Eight of the 16 probands were sporadic cases.

Discussion

Here we describe the largest series of EBS patients with 65 unrelated families being characterized

at the clinical, histological, and DNA level. In 49 families KRT5 and KRT14 mutation analysis

revealed a total of 37 mutations, of which 11 mutations are novel. Our main observations are: 1)

association of mutations in mainly the HBMs, and in particular the K14 HIM, with the most severe

EBS-DM phenotype with keratin clumping in skin; 2) the high percentage of sporadic patients

412Human_K14Human_K15Human_K10Human_DesminHuman_VimentinChimpanzee_K14_predBovine_K14Mouse_K14Rat_K14Chicken_K14

DV KT RL EQ E I AT Y RRD I KT RL EQ E I AT Y RSD I K I RL E NE I Q T Y RSNV KMAL DV E I AT Y RKNV KMAL D I E I AT Y RKDV KT RL EQ E I AT Y RRDV KT RL EQ E I AT Y RRDV KT RL EQ E I AT Y RRDV KT RL EQ E I AT Y RRDV KCRL EQ E I AT Y RR

Human_K5Chimpanzee_K5Bovine_K5Mouse_K5Rat_K5Chicken_K5WesternFrog_K5Human_K3Human_K1Human_DesminHuman_Vimentin

323V S DT S V V L SV S DT S V V L SV S DT S V V L SV S DT S V V L SV S DT S V V L SV S DT S V V L SI S DT S V V L SI S DT S V V L SI S E T NV I L SLQ EQQ VQ V EI Q EQ HVQ I D

199DT KWT L LDT KWT L LDT KWAL LDT KWAL LDT KWT L LE T KWS L LE T KWNL LE T KWNL LQ T KWE L LAAE V NRLL AE L EQ L

149 151V NQ S L L T P L NLV NQ S L L T P L NLV NQ S L L T P L NLV NQ NL L T P L NLV NQ NL L T P L NLV NQ S L L AP L KLV NQ T L L AP L NLI NQ S L LQ P L NVI NQ S L LQ P L NVAG E L L DF S L ADLQ DS L DF S L AD

180 187NNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNKF AS F I DKV RF L EQNNQ F AS F I DKV RF L EQNDRF ANY I E KV RF L EQNDRF ANY I DKV RF L EQ

443LQ KAKQ DMALQ KAKQ DMALQ KAKQ DMALQ KAKQ DMALQ KAKQ DMALQ KAKADL ALQ KCKQ EMALQQ AKDDL ALQQ AKE DL AI RHL KDEMAI Q NMKE EMA

68

Chapter 2

with proven de novo mutations (39%), and 3) the high percentage (25%) of patients with biopsy-

proven EBS without mutations in KRT5 or KRT14.

In the EBS population presented here we found the phenotype of EBS-DM in all but

one patient (with mutation KRT5:p.Ala438Asp) associated with mutations in the HBMs of K5

and K14, in particular the HIM of K14. This finding is in agreement with data obtained from

previously reported KRT5 and KRT14 mutations (figure 2, see the Human Intermediate Filament

Database: www.interfil.org).12 The sequences in these HBMs are highly conserved among other

species and in other intermediate filaments. In vitro studies have shown that these sequences

play an essential role in the initial dimer-dimer interactions in filament formation.26-29 The strong

association of the K14 HIM with EBS-DM is mainly due to a high amount of mutations affecting

“hotspot” codon p.Arg125. This is most likely as a result of the CpG dinucleotide in this codon

which accounts for an unusual high percentage of mutations because of its susceptibility to

methylation and deamination.30, 31 However, milder EBS-loc phenotypes were associated with

missense mutations in HBMs as well, like KRT5:p.Glu170Gly, KRT5:p.Asn177Ser, KRT5:p.Gly476Asp.

The position of the mutation in the local structure, the heptad (abcdefg)n, is thought to influence

the clinical outcome also.32 Heterodimers like K5-K14 are coiled-coil structures with the keratin

filaments interacting through hydrophobic interactions between apolar residues in positions

a and d of the heptad and stabilized by interactions between hydrophilic residues at positions

e and g. It has been suggested that mutations affecting these positions cause the more severe

phenotypes. In our series this was not observed. For example, KRT14 mutations p.Ile412Phe

and p.Leu408Met affect positions a and d of the heptad repeat structure, but both result in

the mild EBS-loc phenotype and mutation p.Glu475Lys involving position b in the heptad-

structure caused severe EBS-DM in another patient (table 1). Of note, mutation p.Gly476Asp

affecting the adjacent amino acid was associated with mild EBS-loc. Most likely the position

of the substituted amino acid in the protein and in the local structure within the protein, in

combination with the physical-chemical nature of the amino acid change, determine the effect

of the mutation on protein functioning, filament assembly, and ultimately the phenotype. In

addition, other factors (environmental, epigenetic, genetic background) may govern the clinical

outcome as well. This is underscored in this study by KRT5 mutations p.Glu170Gly, p.Ala180Asp,

and c.1649delG, being associated with different phenotypes compared to previous reports of

patients with identical mutations, and the intrafamilial variation observed in several families

in this study (table 1). External temperature clearly seems to influence the severity of blistering

exemplified by the reports of majority of patients that blistering is more profound in the summer

months. Additionally, patients noted that rise of body temperature by upcoming illness like

the flu reduced their blister rate, as if the basal cell keratins are stabilized by internal warmth,

or alternatively: the elicited immune reaction against a (viral) infection and the production of

cytokines might have its effects on keratin stability and the formation/breakdown of keratin

aggregates. Recently topical application of sulphoraphane, which is a substance of broccoli,

was shown to prolong survival of K14 -/- mice, probably by upregulation of other keratins.33

69


These findings underscore that keratin cytoskeleton function is a highly dynamic and redundant

system which is interactive with environment.

This study revealed a high rate (39%) of proven de novo mutations meaning both

parents did not carry the mutation. Each mutation was excluded in matched control DNA

samples, hereby minimizing the possibility of the amino acid substitution being a single

nucleotide polymorphism (SNP). Non-paternity may be underlying some of the seemingly de

novo mutations, as is also germline mosaicism in one of the parents.24 Another possibility is that

hardly any changes in the highly conserved keratins are tolerated and that any change results in

disease phenotype. Thus the high percentage de novo events may reflect a high mutability at this

part of the human genome during reproduction. The three codons more prevalently affected

by mutations in multiple families in this study, KRT14:p.Arg125 (n=5 families) KRT14:p.Arg388

(n=3 families) and KRT5:p.Arg187 (n=4 families) contain the highly mutable CpG-dinucleotide

(discussed above).31 Alternatively, the four Dutch families with EBS-loc and mutation KRT5:p.

Arg187Pro might share one common founder.

Similar to the 22% in the EBS population in the United Kingdom19, we found a

percentage of 25% of EBS patients without mutations in the KRT5 or KRT14 genes. The complete

coding sequences of KRT5 and KRT14 were analyzed and we excluded autosomal recessive

null mutations by showing unreduced expression of both proteins by immunofluorescence.

In addition, larger deletions or duplications were excluded by cDNA analysis. Of note, smaller

insertions or deletion will be missed, but these are expected to be found up by the amplification

and sequencing of single exons with their exon-intron borders. K5 or K14 haploinsufficiency

is unlikely as this causes other phenotypes (Dowling Degos Disease [MIM#179850]34, Naegeli

Franceschetti Jadassohn Syndrome [MIM#161000], and Dermatopathia Reticularis Pigmentosa

[MIM#125595]35 (figure 2)). All together this indicates that other genes besides KRT5 and KRT14

may be involved in EBS. The strength of our study lies in the confirmation of intraepidermal

skin fragility in the patient’s skin, hereby providing the definitive diagnosis of EBS. The clinical

differentiation with other hereditary skin fragility diseases with sometimes preferential

hands and feet involvement, like acral peeling skin syndrome [MIM#609796] (mutations in

transglutaminase 5), suprabasal EBS (lethal acantholytic EB due to mutations in desmoplakin

[MIM#609638]; and skin fragility ectodermal dysplasia due to mutations in plakophilin-1

[MIM#604536]), forms of epidermolytic ichthyosis (mutations in K1 and K10 [MIM#113800]),

various forms of pachyonychia congenita (PC1 [MIM#167200], PC2 [MIM#167210], mutations in

K6a, K6b, K16 and K17), and localized non-Herlitz junctional epidermolysis bullosa [MIM#226650]

may be difficult.

Other genes besides KRT5 and KRT14 that have been associated with basal

intraepidermal skin blistering in single patients or families are: PLEC1 encoding the versatile

cytolinker protein plectin which connects the basal keratins to the hemidesmosomal plaque (in

EBS-Ogna [MIM#131950]), COL17A1 encoding the hemidesmosomal protein type XVII collagen,

and ITGB4 encoding the hemidesmosomal integrin β4 protein.36-39 Immunofluorescence

70

Chapter 2

stainings for integrin β4 and type XVII collagen on skin biopsies of EBS patients without KRT5

and KRT14 mutations in this study revealed no abnormalities. However, this does not exclude

mutations in these genes. PLEC1 could be a likely candidate as an autosomal dominant missense

mutation was associated with a non-syndromic EBS phenotype strongly resembling EBS due

to K5/K14 mutations.36 Another hemidesmosomal protein that is located at the cytoplasmic

site of hemidesmosomes and binds to K5 and K14 is BP230. No human disease phenotype

has been associated with mutations in BP230 yet. BP230 -/- mice show skin blistering next

to neurodegenerative disease (BP230 is also expressed in neuronal tissue).40 It is tempting to

speculate that mutations specifically affecting the epidermal isoform of BP230 cause EBS. K15

is another keratin besides K5 and K14 that is expressed in basal keratinocytes, and this protein

may form heterodimers with K5 as well. K15 is thought to partly rescue K14 -/- mice, which

show a less severe phenotype compared to K5 -/- mice. Similarly, in humans K14-null mutations

lead to a relatively mild phenotype, whereas homozygous K5-null mutations have not been

identified. No human disease has been associated with mutations in KRT15 at present. Rugg et

al.19 searched for mutations in KRT15 in their mutation negative EBS cases and Kemp et al.41 did

the same in an EBS patient cohort from Australia and New Zealand, but neither of them found

a mutation in KRT15. Another possible explanation for fragility of the basal keratin skeleton

could be a defective protein involved in keratin assembly, elongation and/or functioning,

like for example proteins influencing the phosphorylation of keratins which in turn affect the

polymerisation status (for review see42, 43). Mutations in desmosomal proteins expressed in the

basal epidermal layers could influence the basal keratin cytoskeleton stability as well. Liovic et

al. recently showed that missense mutations in the helix boundary motifs (associated with the

severe EBS-DM phenotype) cause down-regulation of hemidesmosomal, desmosomal and gap

junction proteins, suggesting close interaction and regulation between the keratin filament

system and the cell junctions.44 Furthermore, recent studies on the autoimmune blistering

disease pemphigus vulgaris indicate that retraction of the cytoskeleton is one of the steps in

acantholysis upon binding of autoantibodies to desmosomal cadherins like desmoglein 3.45

Similarly, mutations in desmoglein 3 or desmocollin 3 could affect basal cell integrity. Alternative

explanations for KRT5 and KRT14 mutation negative EBS could lie in epigenetic phenomena,

like for example paramutation. Paramutation is a process in which genetic instructions can

be passed on to subsequent generations (through small RNAs) without the gene variants

themselves being transmitted.46, 47 In other words, offspring of affected individuals do not carry

the original gDNA mutation but instead show a phenotype due to inherited microRNAs packed

in germ cells. This might go on for several generations. In mice increasing evidence is found for

existence of this phenomenon.47-49 All these data could be subject for future investigations in

patients ascertained of having EBS at the clinical and tissue level but without KRT5 and KRT14

gene mutations.

In conclusion, EBS is known as a hereditary disease of the basal epidermal keratins

K5 and K14 with a discernable phenotype-genotype correlation. Our data implicate that this

71


might be a somehow too simplistic view and we believe that the finding of a high percentage

of EBS patients not carrying mutations in either one of the genes KRT5 and KRT14 warrants

further research. Considering the similar phenotype and mode of inheritance associated with a

previous reported plectin missense mutation we propose PLEC1 as a first candidate.

Acknowledgements

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

to thank Prof. E. Legius (Department of Human Genetics, Catholic University Leuven, Leuven,

Belgium) and Dr. M.A. Morren (Department of Dermatology, Catholic University Leuven, Leuven,

Belgium) for providing DNA samples and clinical data from three patients (BE01, BE02 and BE03)

with EBS. The work was supported by the J.P. Nater Foundation.

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9.

76

Chapter 2

Tabl

e 1.

Phe

noty

pes

and

KRT5

and

KRT

14 g

enot

ypes

of t

he E

BS fa

mili

es d

escr

ibed

in th

is s

tudy

(nov

el m

utat

ions

in b

old)

.

Fam

ilies

Gen

eM

utat

ion

(c.)*

Mut

atio

n (p

.)*D

omai

nH

epta

dO

rigi

nIn

heri

tanc

eEB

S su

btyp

eO

nset

bl

iste

ring

Add

itio

nal c

linic

al

feat

ures

Clin

ical

in

traf

am

vari

abili

ty

EM:

clum

ping

Publ

ishe

d in

**

Phen

otyp

e in

pre

viou

s re

port

s

EB05

7 EB

060

EB11

0KR

T574

C>T

Pro2

5Leu

V1n.

aD

utch

AD

(1x)

, de

nov

o (2

x)M

Pva

riabl

e (b

irth-

6y)

pitt

ed P

PK, t

eeth

ye

sno

50M

P 23

BE03

KRT5

446T

>CLe

u149

Peo

H1

n.a

Belg

ian

AD

eo-lo

cbi

rth

nails

no

no

EB13

5KR

T545

1A>C

Thr1

51Pe

oH

1n.

aD

utch

AD

loc

1 y

none

nono

EB19

0KR

T550

9A>G

Glu

170G

ly1A

, HIM

fM

-Ede

nov

o >

AD

loc

1 y

PPH

nono

ge

n-nD

M 19

EB17

9KR

T553

0A>G

Asn

177A

sn1A

, HIM

fD

utch

AD

loc

varia

ble

(birt

h->1

0 y)

PPH

yes

no

loc

51

EB20

1KR

T553

9C>A

Ala

180A

sp1A

, HIM

bM

-Ede

nov

oge

nbi

rth

apla

sia

cutis

n.a.

no

DM

18

EB20

9KR

T553

8G>C

Ala

180P

ro1A

, HIM

bD

utch

AD

DM

birt

h

PPK

, nai

ls, t

eeth

, co

mpo

und

naev

i, hy

po-+

hy

perp

igm

enta

tion

, ap

lasi

a cu

tis

noye

s

EB08

0KR

T555

6-1G

>CVa

l186

_G

ln18

9del

1An.

aD

utch

de n

ovo

gen

birt

hfo

cal P

PK, n

ails

n.

a.no

14

EB03

8 EB

094

EB13

6 EB

144

KRT5

560G

>CA

rg18

7Pro

1Ab

Dut

chA

D (4

x)lo

c1y

PKno

no

EB21

3KR

T557

2A>C

Gln

191P

ro1A

fKo

rean

unkn

own

gen

unkn

own

PPK

unkn

own

unkn

own

ge

n 52

, 53

EB19

7KR

T559

6A>T

Lys1

99M

et1A

gIn

dia

AD

eo-lo

cbi

rth

PKno

no

BE02

KRT5

968T

>GVa

l323

Gly

L12

n.a

Belg

ian

de n

ovo

loc

unkn

own

PKn.

a.no

EB19

5KR

T598

7C>A

Asn

329L

ysL1

2n.

aFi

nnis

hA

Dlo

c1

yPK

nono

lo

c 54

EB18

2KR

T599

2G>A

Arg

331H

isL1

2n.

aD

utch

AD

loc

1 y

nono

lo

c 32

EB06

3KR

T512

10A

>GLy

s404

Glu

2Be

Dut

chA

Dlo

c1

yno

no14

EB07

3KR

T513

13C>

AA

la43

8Asp

2Bg

Dut

chde

nov

oD

Mbi

rth

PPK

n.a.

yes

14

77


(Tab

le 1

con

tinue

d)

Fam

ilies

Gen

eM

utat

ion

(c.)*

Mut

atio

n (p

.)*D

omai

nH

epta

dO

rigi

nIn

heri

tanc

eEB

S su

btyp

eO

nset

bl

iste

ring

Add

itio

nal c

linic

al

feat

ures

Clin

ical

in

traf

am

vari

abili

ty

EM:

clum

ping

Publ

ishe

d in

**

Phen

otyp

e in

pre

viou

s re

port

s (r

efer

ence

)

EB13

7KR

T513

29G

>CLy

s443

Asn

2Be

Dut

chA

Dlo

c +

eo-lo

cva

riab

lePP

K, P

PHye

sno

EB11

6KR

T514

01C>

GIle

467M

et2B

, HTM

aD

utch

de n

ovo*

**ge

nbi

rth

PPH

, nai

lsye

sno

ge

n-nD

M 55

EB05

4KR

T514

23G

>AG

lu47

5Lys

2B, H

TMb

Dut

chde

nov

o >

AD

DM

birt

hPP

K, n

ails

noye

s14

DM

(Y

asuk

awa,

20

06)

EB16

6KR

T514

27G

>AG

ly47

6Asp

2B, H

TMc

Dut

chA

Dlo

c1

yno

no

loc

56

EB21

8KR

T516

35de

lGLe

u546

S er

fsX8

2V2

n.a

Dut

chde

nov

o >

AD

gen

birt

hPP

K, n

ails

, hyp

o-an

d hy

perp

igm

enta

tion

nono

lo

c 20

BE01

KRT5

1649

delG

Gly

550

Ala

fsX7

7V2

n.a

Belg

ian

AD

MCE

(?)

birt

hM

igra

tory

ves

icle

s on

er

ythe

mat

ano

no

MCE

21, 2

4 M

P 22

EB01

9,

EB05

3,

EB07

5,

EB15

3

KRT1

437

3C>T

Arg

125C

ys1A

, HIM

gD

utch

(3

x), M

-E

(1x)

de n

ovo

(4x)

DM

birt

hPP

K, n

ails

n.a.

yes

D

M 5

EB04

6KR

T14

374G

>CA

rg12

5His

1A, H

IMg

Dut

chA

DD

Mbi

rth

PPK,

nai

lsno

yes

D

M 5

EB16

4KR

T14

383_

385

delT

CCSe

r128

del

1A, H

IMc

Dut

chde

nov

oD

Mbi

rth

PPK,

nai

lsn.

a.ye

s

DM

57

EB10

5KR

T14

389T

>CLe

u130

Pro

1A, H

IMe

M-E

de n

ovo

DM

birt

hPP

K, n

ails

n.a.

yes

14

EB01

4,

EB06

7,

EB07

0KR

T14

526-

2A>C

[Ile1

76

Valfs

X2]

+ [Il

e176

Pr

ofsX

30]

1Bn.

aD

utch

AR

(3x)

AR

birt

hPP

K, g

ener

aliz

ed

hype

rker

atot

ic

plaq

ues

yes

no16

, 17

EB10

8KR

T14

740_

748

del9

ins3

p.A

la24

7_Ly

s250

de

linsG

lu1B

n.a

Dut

chA

Dlo

c1

yno

no

78

Chapter 2(T

able

1 c

ontin

ued)

Fam

ilies

Gen

eM

utat

ion

(c.)*

Mut

atio

n (p

.)*D

omai

nH

epta

dO

rigi

nIn

heri

tanc

eEB

S su

btyp

eO

nset

bl

iste

ring

Add

itio

nal c

linic

al

feat

ures

Clin

ical

in

traf

am

vari

abili

ty

EM:

clum

ping

Publ

ishe

d in

**

Phen

otyp

e in

pre

viou

s re

port

s (r

efer

ence

)

EB09

6KR

T14

927+

1G>T

?L2

/2B

n.a

Dut

chde

nov

o >

AD

eo-lo

cbi

rth

PKye

sno

EB12

8KR

T14

927+

1G>A

?L2

/2B

n.a

Dut

chde

nov

oeo

-loc

birt

hPK

n.a.

no14

loc

25

EB16

0KR

T14

1130

T>A

Ile37

7Asn

2Ba

Dut

chde

nov

olo

c1

yn.

a.no

lo

c 58

EB15

0,

EB15

7,

EB16

5KR

T14

1162

C>T

Arg

388C

ys2B

eD

utch

AD

(3x)

loc

+ eo

-locva

riabe

l (b

irth

- 2 y

)PK

, nai

lsye

sno

lo

c 19

, 58

EB09

5KR

T14

1222

C>A

Leu4

08M

et2B

dD

utch

de n

ovo

loc

3 y

nails

n.a.

no14

EB11

5KR

T14

1231

_123

3 de

lGAG

Glu

411d

el2B

gD

utch

de n

ovo

> A

Dlo

c1

yno

neno

no

loc

32

EB16

7KR

T14

1234

A>T

Ile41

2Phe

2Ba

M-E

AD

loc

3 y

PPK

nono

EB20

5KR

T14

1240

-12

49de

l10

Thr4

14

Ala

fsX2

52B

, HTM

an

d ta

iln.

a.D

utch

De

novo

gen

birt

hA

plas

ia c

utis

n.a.

no

EB06

9KR

T14

1256

T>A

Leu4

19G

ln2B

, HTM

aD

utch

de n

ovo

> A

DD

Mbi

rth

PPK,

nai

lsye

sye

s14

, 59

* N

ucle

otid

e nu

mbe

ring:

+1

corr

espo

nds

to th

e A

of t

he A

TG tr

ansl

atio

n in

itiat

ion

codo

n in

the

refe

renc

e se

quen

ce (R

efSe

qs K

RT5:

NM

_000

423.

5, K

RT14

: NM

_000

526.

3)**

Pub

lishe

d in

: som

e of

the

patie

nts/

fam

ilies

in th

is s

tudy

hav

e be

en p

revi

ousl

y re

port

ed, r

efer

ence

is g

iven

*** T

wo

affec

ted

sist

ers

with

una

ffect

ed p

aren

ts n

ot c

arry

ing

the

mut

atio

n, p

ossi

bly

germ

line

mos

aici

smA

D, a

utos

omal

dom

inan

t; M

P, m

ottle

d pi

gmen

tatio

n; D

M, D

owlin

g-M

eara

; EBS

, epi

derm

olys

is b

ullo

sa s

impl

ex; E

M, e

lect

ron

mic

rosc

opy;

gen

, gen

eral

ized

non

-Dow

ling-

Mea

ra; H

IM, h

elix

in

itiat

ion

mot

if; H

TM, h

elix

ter

min

atio

n m

otif;

loc,

loca

lized

; nai

ls, n

ail a

bnor

mal

ities

; PPH

, pal

mop

lant

ar h

yper

hidr

osis

; PK,

pla

ntar

ker

atod

erm

a; P

PK, p

alm

opla

ntar

ker

atod

erm

a; M

CE,

mig

rato

ry c

ircin

ary

eryt

hem

a; M

-E, M

iddl

e-Ea

st c

ount

ries;

y, y

ear;

n.a.

, not

app

licab

le; t

eeth

, tee

th a

bnor

mal

ities

.

university of groningen epidermolysis bullosa simplex ... · 59 krt5 and krt14 mutations in 75% of...

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