ichthyosis vulgaris: novel flg mutations in the german population and high presence of cd1a+ cells...

CLINICAL AND LABORATORY INVESTIGATIONS BJD British Journal of Dermatology

Ichthyosis vulgaris: novel FLG mutations in the Germanpopulation and high presence of CD1a+ cells in theepidermis of the atopic subgroupV. Oji, N. Seller, A. Sandilands,* R. Gruber,� J. Gerß,� U. Huffmeier,§ H. Hamm,– S. Emmert,** K. Aufenvenne,D. Metze, T. Luger, K. Loser, I. Hausser,�� H. Traupe and W.H.I. McLean*

Department of Dermatology, University of Munster, Von-Esmarch-Str. 58, 48149 Munster, Germany

*College of Life Sciences, Epithelial Genetics Group, Division of Molecular Medicine, University of Dundee, College of Life Sciences, Dundee, U.K.

�Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria

�Department of Medical Informatics and Biomathematics, Interdisciplinary Centre Of Clinical Research, University of Munster, Germany

§Institute of Human Genetics, University Hospital Erlangen, University Erlangen-Nurnberg, Erlangen, Germany

–Department of Dermatology, University of Wurzburg, Wurzburg, Germany

**Department of Dermatology, Georg August University, Gottingen, Germany

��Department of Dermatology, University of Heidelberg, Heidelberg, Germany

CorrespondenceVinzenz Oji.

E-mail: [email protected],

[email protected]

Accepted for publication9 October 2008

Key wordsdendritic cells, diagnostics, epidermal barrier,

genetics, keratinisation disorders

Conflicts of interestNone declared.

V. Oji and N. Seller contributed equally.

DOI 10.1111/j.1365-2133.2008.08999.x

Summary

Background Ichthyosis vulgaris (IV) is a genetic disorder with a prevalence of1 : 250–1000 caused by filaggrin (FLG) mutations, which also predispose toatopic diseases.Objectives To study the genotype ⁄phenotype relationship in IV and to analysewhether the suggested skin barrier defect is associated with differences of epider-mal dendritic cells.Patients ⁄methods We evaluated a cohort of 26 German patients with IV, establishedan IV severity score and analysed epidermal ultrastructure, histology, filaggrinand CD1a antigens. Mutations were screened by restriction enzyme analysis. Par-ticular sequencing techniques allowed the complete FLG analysis to reveal novelmutations.Results The combined null allele frequency of R501X and 2282del4 was 67Æ3%.Patients also showed the mutations S3247X and R2447X as well as five novel FLGmutations: 424del17 and 621del4 (profilaggrin S100 domain), 2974delGA(repeat 2), R3766X (repeat 101) and E4265X (repeat 102). Their combined allelefrequency in controls was < 0Æ7%. No mutation was found in one IV patient, allin all ~27% were heterozygous, and the majority (~69%) showed two nullalleles. The IV severity score and ultrastructure showed a significant correlationwith genotypes. Interestingly, CD1a cell counts showed a significant differencebetween nonatopic and atopic IV patients both with eczema and without eczema.Conclusions We confirm that the mutations R501X and 2282del4 represent themost frequent genetic cause in German IV patients. The novel mutations areprobably population and family specific. The observed differences of CD1a cellssupport the hypothesis that there is a barrier defect that predisposes to atopicmanifestations, possibly independent of atopic eczema.

Ichthyosis vulgaris (IV) (OMIM 146700) has an estimated prev-

alence of 1 : 250–1000 and is the most common form of ich-

thyosis1 characterized clinically by light grey scaling, keratosis

pilaris, increased palmoplantar markings and associated atopic

manifestations.2,3 The histology of IV epidermis is characterized

by mild orthohyperkeratosis and a reduced or absent stratum

granulosum (SG).1,4 However, the SG can be normal in some

cases even in patients with clinically pronounced IV.5,6 The

ultrastructural hallmark of IV is represented by abnormally low

numbers of often crumbly keratohyaline (KH) granules.7

In 2006, it was shown that common filaggrin (FLG) null

mutations are the molecular cause of the disorder.8 The FLG

� 2009 The Authors

Journal Compilation � 2009 British Association of Dermatologists • British Journal of Dermatology 2009 160, pp771–781 771

gene product profilaggrin encompasses ~324 amino acids and

is initially synthesised as a 435 kDa insoluble, phosphorylated

precursor containing 10–12 highly repetitive polymorphic full

tandem repeats.9 It constitutes one of the major components

of the KH granules.10 During differentiation profilaggrin is

dephosphorylated and proteolytically cleaved. Effective and

complete sequencing of the entire FLG gene is a challenge.

Overlapping polymerase chain reaction (PCR) and unidirec-

tional deletion strategies, described recently,11 have revealed

more than 20 different FLG null mutations in different

populations so far.8,12–18 The most common nonsense

mutations R501X and 2282del4 are located in repeat 1 near

the N-terminus.8

Most interestingly, a strong association between FLG muta-

tions and atopic eczema (AE) has been established19 and repli-

cated in several populations.11,12,15–23 Thus the potential of IV

as a model that allows an understanding of how a defect of

the epidermal barrier predisposes to common atopic diseases

is most appreciated.24,25 However, only a few studies, in

which IV patients were recruited independently from atopic

disorders, have linked the molecular findings with clinical and

ultrastructural investigations so far.8,13,14

Therefore, we decided to evaluate a large cohort of German

patients with IV, investigate the presence of filaggrin muta-

tions by complete FLG sequencing analysis and correlate the

clinical findings and those of the histology, ultrastructure and

immunohistochemistry. Moreover, we studied the expression

of CD1a+ cells in a number of skin biopsies of patients with

IV with and without atopic diseases in comparison with nor-

mal skin. Our findings indicate that changes in the epidermal

dendritic cell network are present in IV with concomitant ato-

pic diseases.

Materials and methods

Patients

This study was approved by the institutional review board of

the University Hospital of Munster; all patients enrolled gave

their informed consent. The diagnosis was made by two expe-

rienced dermatologists specialized in cornification disorders

(H.T. and V.O.). In males X-linked recessive ichthyosis was

excluded by testing for steroid sulfatase activity when neces-

sary. Patients were recruited from independent families from

the network for ichthyosis and related keratinization disorders

NIRK (http://www.netzwerk-ichthyose.de, accessed 3 Jan

2009) and were primarily consulting the dermatologists

because of their ichthyosis (Fig. 1). All patients were ques-

tioned about a history of eczema, allergic rhinoconjunctivitis

and asthma. The diagnosis of AE was made according to Hani-

fin and Rajka’s criteria for atopic dermatitis.26 The IgE level

was determined to confirm the diagnosis of allergic rhinocon-

junctivitis. Punch biopsies (1–3 · 4 mm) were obtained from

17 of the 26 patients with IV and from 10 healthy controls.

Usually the buttocks were chosen as the biopsy site.

Ichthyosis vulgaris severity score

For this study we developed an IV severity score based on the

following criteria: severity of scaling (absent = 0, mild = 1,

moderate = 2, marked = 3), colour of scales (not applica-

ble = 0, light grey–white = 1, grey ⁄brown ⁄dark grey = 2),

palmoplantar markings (uncertain = 0, evident ⁄present =

1, marked = 2), hypohidrosis (as noted by patients: lack-

ing = 0, present = 2), keratosis pilaris [absent = 0, mild

(a) (b)

Fig 1. Clinical presentation of ichthyosis vulgaris due to the compound heterozygous genotype R501X ⁄2282del4 (patient ID 22). The young lady

working as an apprentice in a bakery showed pronounced palmoplantar markings (a) and suffered from moderate light grey scaling mainly on the

trunk (b). She never experienced atopic eczema, but reported seasonal rhinoconjunctivitis. We confirmed an elevated IgE of 1850 kU L)1 with

high specific IgE to wheat flour and house dust mite.


Journal Compilation � 2009 British Association of Dermatologists • British Journal of Dermatology 2009 160, pp771–781

772 Novel FLG mutations and elevated CD1a+ cell counts in ichthyosis vulgaris, V. Oji et al.

(upper arm) = 1, severe (+ trunk or distal extremities) = 2],

and family history of dry skin, scaling or palmoplantar

markings (negative = 0, positive in one parent or in sib-

lings = 1, positive in both parents = 2). Raw data were taken

from the NIRK clinical findings form (available at http://

www.netzwerk-ichthyose.de/fileadmin/nirk/uploads/Erfassungs

bogen_englisch.pdf last accessed 16 November 2008). A

Mann–Whitney test was performed for the calculation of the

IV severity score differences.

Genetic analysis

Genomic DNA was extracted from peripheral blood leuco-

cytes using standard procedures. For patients with IV the mu-

tations R501X and 2282del4 were screened using the DraIII

and NlaIII restriction enzyme sites as described previously.8,14

The R2447X mutation was screened by digestion of PCR

products with NlaIII (Hin1II) for 6 h at 37 �C. Genomic

DNA was amplified using Taq polymerase mix (Promega,

Mannheim, Germany) and the following primers: forward

5¢-ACG TGG CCG GTC AGC A-3¢ and reverse 5¢-CCT GAC

CCT CTT GGG ACG T-3¢. The PCR reaction was performed in

35 cycles of 94 �C for 5 min, 35 cycles of 94 �C for 40 s,

58 �C for 50 s, 72 �C for 1 min. Digests were run on 3%

(w ⁄v) agarose gels and ⁄or 10% polyacrylamide ⁄TBE gels.19,27

Data for the 376 control probands refer to the same study

group as in Huffmeier et al.27 A v2 test was calculated to

compare allele frequencies. If the number of expected obser-

vations was low, Fisher’s exact test was used.

Patients’ DNA that was negative or heterozygous for one of

the three pre-screened mutations was fully sequenced using

primers and conditions as reported previously.11 The PCR

products were sequenced on an ABI PRISM 3730 genetic ana-

lyser (Applied Biosystems, Foster City, CA, U.S.A.). The preva-

lent mutation S3247X was confirmed using the TagMan

assay.11 To determine allelic differences in patients with new

frameshift mutations, PCR products were cloned into a pCR

2.1 vector (Invitrogen, Karlsruhe, Germany) (Fig. S1). Plasmid

DNA was linearized by restriction enzyme digestion and

sequenced using M13 forward and reverse primers. The newly

identified mutations 424del17, 621del4 and 2974delGA were

screened by sizing of fluorescent labelled PCR products

(Fig. S2). PCR conditions and product sizes are given in

Table S1. R3766X was screened using restriction enzyme anal-

ysis. A 322 bp PCR product was amplified in a 25 lL reaction

volume containing 1Æ5 mmol L)1 MgCl2, 10 pmol forward

primer 5¢-GCCCATGGGCGGACCAGGA-3¢ and reverse primer

5¢-GCTTCATGGTGATGCGACCA-3¢, 0Æ25 mmol L)1of each of

dNTP, 4% DMSO, and 1 U high-fidelity polymerase mix

(Roche, Penzberg, Germany). The PCR amplification condi-

tions were as follows: one cycle of 94 �C (5 min) followed

by 35 cycles of 94 �C (30 s), 62 �C (30 s), 72 �C (45 s), and

a final extension cycle of 72 �C (5 min). A 5 lL volume

of PCR product was incubated with 0Æ25 U of AluI (New

England Biolabs, Frankfurt am Main, Germany) in a 20 lL

reaction volume at 37 �C overnight and the mutant allele was

detected with 202 bp, 70 bp and 60 bp fragments on a 3%

agarose gel. The wild type allele showed fragments of 262

and 70 bp. The novel mutation E4265X was screened using

restriction enzyme digestion with MseI: a 398 bp PCR pro-

duct was amplified in 25 lL reaction volume containing

1Æ5 mmol L)1 MgCl2, 10 pmol forward primer 5¢-GATGGCT-

CCAGACACCCCAG-3¢ and reverse primer 5¢-CTCCAGTACTGG-

GCCCAGC-3¢, and 0Æ25 mmol L)1 each of dNTP, 4% DMSO,

and 1 U high-fidelity polymerase mix (Roche). A 5 lL

volume of PCR product was incubated with 0Æ5 U of MseI

(New England Biolabs) in a 20 lL reaction volume at 37 �Cfor 2 h and detected on a 2% agarose gel showing fragments

of 350 bp and 48 bp, whereas the wild-type allele had no

restriction site for MseI (Fig. S1).

Ultrastructural analysis

All specimens were fixed for at least 2 h at room tempera-

ture (RT) in 3% glutaraldehyde solution in 0Æ1 cacodylate

buffer pH 7Æ4, cut into pieces of approximately 1 mm3,

washed in buffer, post-fixed for 1 h at 4 �C in 1% osmium

tetroxide, rinsed in water, dehydrated through graded etha-

nol solutions, transferred in propylene oxide, and embedded

in epoxy resin (glycidether 100). Semithin and ultrathin sec-

tions were cut with an ultramicrotome (Reichert Ultracut E,

Depew, NY, U.S.A.). Semithin sections were stained with

methylene blue. Ultrathin sections were treated with uranyl

acetate and lead citrate, and examined with an electron

microscope (Philips EM 400, Hamburg, Germany).

Immunohistochemistry

Unfixed cryosections were blocked with normal goat serum

(Jackson ImmunoResearch Laboratories Inc., West Grove, PA,

U.S.A.) in PBS for 30 min at RT, and then incubated for 16 h

at RT with mouse IgG1 kappa anti-filaggrin (clone 15C10)

1 : 100 in PBS, which corresponds to a portion of the

N-terminus of the human filaggrin molecule (Novocastra Lab-

oratories Ltd, Newcastle, U.K.). Dendritic cells (DC) were

detected with mouse anti-CD1a 1 : 200 in PBS (Acris Anti-

bodies, Hiddenhausen, Germany). Primary antibodies were

visualized with FITC-goat anti-mouse IgG (Jackson Immuno-

Research Laboratories Inc., West Grove, PA, U.S.A.) diluted

1 : 100 in PBS. Slides were viewed with an Axioscope 2 and

digital images taken using an Axiocam HR video camera and

Axiovision 3.0 software (all Carl Zeiss, Jena, Germany). DCs

were localized in the epidermis from the basal layer upwards

analysing 0Æ5 mm of the epidermal length for each count. For

each patient 6–9 independent CD1a+ cell counts in four

different skin sections were performed to calculate the mean

value (CD1a+ cells per 0Æ5 mm) and standard deviation. Ten

skin sections of healthy nonatopic skin served as control. The

numbers of epidermal CD1a+ cells were evaluated using a

log-linear mixed model with Poisson variance function. All

statistical analyses were performed using the SAS software

(version 9.1; SAS Institute, Cary, NC, U.S.A.).



Novel FLG mutations and elevated CD1a+ cell counts in ichthyosis vulgaris, V. Oji et al. 773

Results

Genetic findings

The combined risk allele frequency of R501X and 2282del4 was

67Æ3% (Tables 1, 2) giving a highly significant difference com-

pared with healthy controls (P < 5 · 10)5). The complete FLG

sequencing analysis revealed five novel FLG mutations (Figs 2,

3, S1, S2) with low frequency in the control population

(Table 3). All in all, the complete FLG analysis confirmed a

genotype with two null alleles in 18 patients (69Æ2%), one

mutation in seven patients (26Æ9%), and one female patient did

not show any mutation (3Æ9%). One patient harboured the

prevalent null allele R2447X, two patients the recently described

mutation S3247X, which occurred in a mildly affected nona-

topic heterozygous patient and in an atopic compound hetero-

zygous R501X ⁄S3247X patient. Patients with novel mutations

were: (i) index patient 3. This atopic IV patient revealed the

compound heterozygous genotype 2282del4 ⁄424del17. The

latter novel mutation leads to a frameshift within the S100

domain of profilaggrin resulting in a premature termination

codon (PTC) after five codons. The screening by fluorescent

Table 1 Frequency of the two common nullmutations R501X and 2282del4

Mutation

Study group

(number of alleles)

Number ofrisk alleles

(frequency in %)

P-value(Fisher’s

exact test)

R501X Controls (n = 752)* 13 (1Æ8) < 0Æ00005IV group (n = 52) 18 (34Æ6)

2282del4 Controls (n = 752)* 20 (2Æ8) < 0Æ00005IV group (n = 52) 17 (32Æ7)

Combined risk allele frequencyR501X ⁄2282del4

Controls (n = 752)* 33 (4Æ6) < 0Æ00005Entire IV group (n = 52) 35 (67Æ3)

Genotyping rate > 95Æ0%

*Ten healthy volunteers.

Table 2 Study group of 26 individuals with ichthyosis vulgaris: filaggrin genotypes and clinical data including severity of ichthyosis and thepresence of concomitant atopic disorders

ID Age (years) ⁄ sexPalmoplantarmarkings

Ichthyosis severityscore (0–15)* Atopic manifestations FLG genotype

1 30 ⁄ female + 5 Ecz, Rhi, Asth, high IgE R501X ⁄wt

2 27 ⁄ female + 5 None S3247X ⁄wt3 25 ⁄ female ++ 9 Rhi, high IgE 2282del4 ⁄424del17

4 17 ⁄male ++ 7 Ecz 2282del4 ⁄2282del45 51 ⁄ female + 4 Ecz wt ⁄2282del4

6 19 ⁄ female ++ 7 Ecz R501X ⁄R501X7 46 ⁄male + 5 Rhi R501X ⁄wt

8 13 ⁄male ++ 7 Rhi R501X ⁄2282del49 8 ⁄male ++ 12 Ecz, high IgE R501X ⁄2282del4

10 4 ⁄male ++ 7 Ecz R501X ⁄wt11 63 ⁄male ? 6 None R501X ⁄R501X

12 9 ⁄ female ++ 7 Ecz 621del4 ⁄2282del413 53 ⁄male ++ 7 None R501X ⁄R501X

14 52 ⁄male ++ 8 High IgE wt ⁄2282del4

15 12 ⁄male ++ 6 Ecz, high IgE 2974delGA ⁄E4265X16 16 ⁄ female ++ 7 Ecz, high IgE R501X ⁄S3247X

17 26 ⁄ female ++ 7 Ecz R501X ⁄R3766X18 16 ⁄ female + 6 None 2282del4 ⁄wt

19 49 ⁄ female ++ 8 None 2282del4 ⁄2282del420 4 ⁄ female ++ 9 None R501X ⁄2282del4

21 9 ⁄ female ++ 6 High IgE R2447X ⁄2282del422 16 ⁄ female + 9 Rhi, high IgE R501X ⁄2282del4

23 51 ⁄ female ++ 8 None wt ⁄wt24 9 ⁄ female ++ 8 None R501X ⁄2282del4

25 7 ⁄male ++ 9 Ecz, Rhi, high IgE, Asth 2282del4 ⁄2282del426 16 ⁄ female ++ 10 None R501X ⁄R501X

*Refer to Materials and methods.




PCR in 149 healthy German control individuals showed a low

risk allele frequency of 0Æ34%; (ii) index patient 12. The com-

plete FLG analysis of this female IV patient with history of

eczema revealed a second novel FLG null allele, which is also

localized in the S100 domain. The deletion 621del4 results

into a PTC after 15 codons and was not present in the healthy

Mutation R3766X Mutation E4265X

g

M s

gaa→taa

Mu

tati

on

seq

uen

ceW

ild t

ype

seq

uen

ce

aga→tga

Fig 2. Overview of the new nonsense

mutations resulting in a direct stop codon.

Patient 17 showed the heterozygous mutation

R3766X (first column). Patient 15, who was

heterozygous for the two length variants

FLG11 and FLG12, showed the compound

heterozygous genotype 2974delGA ⁄E4265X.

The second mutation E4265X is located

within repeat 102. Repeat 101 and 102 were

cloned into the pCR 2.1 vector and the

separated DNA was subjected to sequence

analysis (column 2, above).

Table 3 Novel FLG mutations and frequenciesin the German control population

Mutation Repeat

Study group

(number of alleles)

Number ofrisk alleles

(frequency in %)

P-value(Fisher’s

exact test)

424del17 First partialrepeat

Controls (n = 298) 1 (0Æ34) 0Æ27IV group (n = 52) 1 (1Æ92)

621del4 First partialrepeat

Controls (n = 322) 0 (0) 0Æ14IV group (n = 52) 1 (1Æ92)

2974delGA 2 Controls (n = 342) 0 (0) 0Æ13IV group (n = 52) 1 (1Æ92)

R3766X 101 Controls (n = 290) 0 (0) 0Æ15IV group (n = 52) 1 (1Æ92)

E4265X 102 Controls (n = 290) 1 (0Æ34) 0Æ28IV group (n = 52) 1 (1Æ92)

1 2 3 7 81 82 9 101 102

R24S

X621del4

424del17

R501X 2282del4

R3766X E4265X2974delGA

47X3247

3321delA

3702delG

R1474X

5360delG

6867delAG

E2422X

7267delCA

S2554X

S2889X

Q3683X

11029delCA

1249insG

S3296X

11033del4

R4307X7945delA

441delA

Q2147X

654

Fig 3. Overview of the locations of all FLG mutations. Mutations in red were present in our patients. Novel mutations are given in bold. Black

mutations published so far and shown below were not present in our study. The yellow boxes show the location of the common null-alleles

R501X and 2282del4.




controls (n = 166); (iii) index patient 15. This boy with an ato-

pic diathesis and eczema showed the compound heterozygous

genotype 2974delGA ⁄E4265X. The first deletion leading to a

frameshift with PTC after 34 codons was located in repeat 2 and

has not been found in the healthy controls (n = 171). The direct

PTC E4265X is due to the substitution c.12793G>T and is

located in FLG repeat 102. Its risk allele frequency in healthy

individuals was 0Æ34%; and (iv) index patient 17. This patient

with IV showed the second mutation R3766X. The novel PTC

mutation located in repeat 101 directly results from the substi-

tution c.11296A>T and has not been observed in the healthy

controls (n = 145).

Clinical correlation

The patients’ ages ranged from four to 63 years (mean age

~25 years). Increased palmoplantar markings were evident in

all patients except one (Table 2). The IV severity score ranging

from 5 to 12 showed a mean value of 7Æ3 in all patients with

IV. Those patients having one FLG mutation had a mean score

of 5Æ1, in contrast to patients with two mutations, who

showed a mean score of 7Æ8 (P = 0Æ0053). Concomitant

atopic diseases in general were present in 65% of the cases.

The frequency of atopic disorders in patients with IV with one

FLG mutation was 71Æ4%, and the atopy frequency in the

double null allele carriers was 66Æ7%. Allergic rhinitis or a

high IgE level were present in 42%, six of these IV patients

showed a history of eczema.

Histology, ultrastructure and filaggrin antigen mapping

Skin biopsies were taken in 17 patients of the study group

(Table 4). Representative examples of patients are shown in

Figure 4. At the histological level the SG was changed in all IV

skin sections, also in the patient without any FLG mutation (ID

23). Patients with two null alleles often showed absent or

strongly reduced SG, while heterozygous patients most often

showed residual or focally normal SG. Similarly, the antigen

mapping showed a reduced but clear-cut immunofluorescence

signal in heterozygous patients and mostly an absent or strongly

reduced signal in the skin of those with a double mutation.

Patient 23 without any FLG mutation had a normal filaggrin

signal. The correlation of the histological SG and filaggrin anti-

gen with the risk allele numbers was not perfectly consistent,

Table 4 Correlation between genotypes and histology, ultrastructure and filaggrin antigen mapping

FLG genotypes in patients with

ichthyosis vulgaris ID

Stratum granulosum

(histology)

Keratohyaline

(electron microscopy)

Filaggrin

antigen (IF)

One null-allele R501X ⁄wt 7 Reduced n.d. n.d.10 Reduced n.d. Reduced

S3247X ⁄wt 2 Reduced, focally normal Minimal, focally normal Reduced2282del4 ⁄wt 5 Reduced Minimal, focally normal n.d.

14 Absent Minimal, focally normal Reduced18 Absent, focally reduced n.d. n.d.

Two null-alleles 2282del4 ⁄2282del4 4 Absent n.d. n.d.19 Absent Minimal crumbly Absent

25 Strongly reduced Minimal crumbly ReducedR501X ⁄R501X 11 Strongly reduced Minimal crumbly Absent

R501X ⁄2282del4 8 Strongly reduced Minimal crumbly Absent9 Strongly reduced Minimal crumbly Absent

R501X ⁄S3247X 16 Focally reduced or absent n.d. Reduced2282del4 ⁄621del4 12 Strongly reduced n.d. Absent

2282del4 ⁄424del17 3 Strongly reduced Minimal crumbly Strongly reduced2974delGA ⁄E4265X 15 Reduced n.d. n.d.

No mutation wt ⁄wt 23 Reduced n.d. Normal

IF, immunofluorescence; n.d., not done; wt, wild type.

Fig 4. Comparison of the histological (column 1), ultrastructural (column 2) and immunofluorescence findings (column 3) of normal skin (a–c),

ichthyosis vulgaris skin with heterozygous mutation S3247X (d–f, patient ID 2), and ichthyosis vulgaris skin of two double null allele carriers

(g–l, patient ID 3 and 8). Routine histology shows reduced or minimal stratum granulosum suggesting a lack of keratohyalin in all ichthyosis

vulgaris patients (d, g, j). Electron microscopy visualizes a striking reduction of keratohyalin granules in all double null-allele carriers (h, k).

The heterozygous patient 2 shows minimal, but focally normal keratohyalin structures (black arrows). Compared with normal skin, immuno-

fluorescence of the N-terminal filaggrin antibody demonstrates a clearly reduced signal in the heterozygous patient 2 (f), a marked reduction

of the signal in the compound heterozygous patient 3 (i) and a complete lack of fluorescence in patient 8 (l). N, nucleus; KH, keratohyaline;

SG, stratum granulosum; SC, stratum corneum.




2282del4 /424del17(patient 3)

20 µm3030 µµmm 22 µµmm

N SGSG

SCSCSGSG

SCSCSG

SC

wt/wt(healthy control)

S3247X/wt(patient 2)

R501X/2282del4(patient 8)

Haematoxylin–eosin

Histology FilaggrinUltrastructure

(a) (b) (c)

(f)(e)(d)

(g) (h)

(k)(j)

(i)

(l)




e.g. in patients 16 and 25. In contrast, the ultrastructure assessed

by electron microscopy revealed a clear-cut correlation of geno-

type and KH morphology and demonstrated that there is still a

significant portion of residual KH in all patients (Fig. 4). It was

‘minimally crumbly’ in all double null allele carriers but ‘mini-

mal and focally normal’ in all heterozygous patients with IV.

Differences of CD1a+ cells in nonatopic and atopic skin

with ichthyosis vulgaris

In cases of concomitant eczema, biopsies were taken from

nonlesional skin exhibiting ichthyotic scaling but not eczema.

CD1a is the main DC marker present on Langerhans cells (LC)

and inflammatory epidermal dendritic cells (IEDC).28 Posi-

tively stained cells were counted over an epidermal length of

0Æ5 mm. Ten biopsies of healthy individuals served as controls

and showed an average number of 10Æ5 cells per 0Æ5 mm

(Fig. 5). Patients with IV without any atopic manifestation

showed a similar number of 9Æ4 cells. Patients reporting aller-

gic rhinoconjunctivitis and ⁄or asthma or having an elevated

IgE level (> 200Æ0 kU L)1) but who never suffered from

eczema had an increased rate of 14Æ3 cells per 0Æ5 mm.

Patients with atopic IV with a history of eczema showed 15Æ3CD1a+ cells per 0Æ5 mm. The difference of CD1a+ cell counts

between nonatopic and atopic IV patients was significant

for both atopy subgroups, atopic patients without eczema

(P = 0Æ01) and patients with atopy and eczema (P = 0Æ004).

The comparison of mean CD1a+ numbers of patients with

atopic IV with one mutation and those of patients with atopy

with two mutations showed a nonsignificant difference (10Æ9CD1a+ cells per 0Æ5 mm vs. 12Æ2 CD1a+ cells per 0Æ5 mm).

Discussion

Our genetic study confirms that the FLG null alleles R501X

and 2282del4, first described in 2006,8 have a high com-

bined risk allele frequency (67Æ3%) in German IV patients.

The combined allele frequency in 376 healthy German con-

trols was 4Æ6%, with 2282del4 being more common than

R501X (Table 1), and corresponded to that of the Irish, Scot-

tish, European American and Austrian population screen-

ing.8,14 The less common variants R2447X and S3247X were

only observed in one and two patients, respectively (Table 2).

The FLG gene consists of 10–12 almost identical repeats.9 The

different repeat number is due to variable duplication(s) of

repeat 8 and ⁄or 10. It is therefore technically very demanding

to perform a reliable sequencing of the entire gene.11 After

the first screening we performed a complete FLG sequencing

analysis, which revealed the two novel PTC mutations

E4265X and R3766X (Fig. 2) and the three novel frameshift

mutations 424del17, 621del4 and 2974delGA (Fig. S1). Their

combined allele frequency in the German control group was

extremely low (< 0Æ7%). It is of note that about 1 ⁄4 of the

cases showed only one FLG null allele and that one female

patient (ID 23), who clinically showed IV without atopic

manifestations and had a reduced SG, did not reveal any FLG

Normal skin

IV-patient 16

100 µm

100 µm

(a)

0

2

4

6

8

10

12

14

16

18

20

Atopic IVwith

eczema

Atopic IVwithouteczema

Non-atopicIV

Normalskin

CD1a: cell per 0·5 mm epidermis

n = 4n = 5

n = 4n = 10

(b)

Fig 5. Staining of the prototypic dendritic cell marker CD1a in normal

skin and ichthyosis vulgaris epidermis (a) and CD1a+ cells counted in

the epidermis of patients with ichthyosis vulgaris without atopy, with

atopy but without eczema or with atopy and eczema (b). Epidermal

CD1a signals indicating the presence of dendritic cells (white arrows)

were counted in skin sections of healthy individuals and patients with

ichthyosis vulgaris (a). In normal skin epidermal sectors of 0Æ5 mm

length showed ~10 (± 2) cells. Patients with ichthyosis vulgaris

without atopy had similar CD1a+ counts, but patients with ichthyosis

vulgaris with concomitant atopic manifestations, especially eczema,

displayed significantly higher numbers of CD1a+ cells (P < 0Æ01) (b).




mutation. It therefore prompts us to speculate that there are

other genes involved in the pathogenesis of IV implying

genetic heterogeneity, e.g. in the Chinese population another

locus for IV has been assigned to chromosome 10q22.29 FLG

analysis of Singaporean Chinese IV patients revealed the

presence of the null alleles 441delA, 1249insG, 7945delA,

Q2147X and E2422X and patients with atopy in Japan

showed the two nonsense mutations S2889X and S3296X

(Fig. 3). None of these apparently population specific muta-

tions were observed in the present study suggesting that the

novel mutations from our German cohort are also population

or family specific. Further studies are necessary to analyse

the prevalence of the novel mutations in a large cohort with

atopic disorders.

Clinical and morphological findings correlated

with genotypes

This study concerns a group of 26 carefully characterized

independent German individuals with IV, who attended a spe-

cialized outpatient clinic for disorders of keratinization, and

thus had no recruitment bias for atopic disease. Increased

palmoplantar markings were present in all patients except one,

who additionally suffered from a severe nonepidermolytic

palmoplantar keratoderma (ID 11), and were considered as

part of the IV phenotype rather than attributed to concomitant

atopic dermatitis.30,31 Severe hypohidrosis, appearing as a so

far underestimated clinical finding of IV, and keratosis pilaris30

were observed in many patients with IV and were therefore

included in the IV severity score (see above). Interestingly, an

IV severity score £ 5Æ0 implied that only one FLG mutation

may be found in that patient (Table 2), suggesting that this

score may be used as an excellent clinical screening method.

However, the reliability of the tool has to be confirmed in a

replication cohort.

The antigen mapping performed with the antibody directed

against the filaggrin N-terminus showed a reduced but clear-

cut immunofluorescence signal in heterozygous patients and

mostly an absent or strongly reduced signal in skin with a

double mutation. However, only electron microscopy pro-

duced absolutely consistent results (Table 4). It is of note that

even double null mutation carriers showed at least ‘minimal

and crumbly’ KH. This observation is in line with the notion

that at the electron microscopic level KH consists of different

compartments especially in ridged skin and in the epidermal

duct cells of sweat glands,7 of which only one compartment is

associated with the filaggrin molecules. Moreover, one has to

consider that focal higher amounts of KH in the skin of

patients with IV can be due to proximity to a hair follicle or

sweat gland, where physiologically more KH is found in com-

parison with interfollicular skin. From the results of this large

ultrastructural study, which was combined with a complete

FLG sequencing analysis, we conclude that electron microscopy

represents the most reliable diagnostic tool for patients with

IV and may even be regarded as an equivalent of a complete

genetic analysis.

Different numbers of epidermal CD1a+ cells in the skin

of nonatopic and atopic patients with ichthyosis vulgaris

A recent report that showed that filaggrin deficiency leads to

reduced levels of the natural moisturizing factor (NMF)32 sup-

ports the important role of an impaired epidermal barrier in

the development of eczema and ⁄or other atopic manifesta-

tions. Atopic disorders in general were present in ~2 ⁄3 of the

cohort of this study. Allergic rhinitis or a high IgE level were

present in 42%, but only six of these patients with IV reported

a history of eczema. This observation suggests that allergic

sensitization in FLG deficiency may develop apart from eczema

and confirms a recent report, which showed that FLG muta-

tions confer a substantial risk for allergic rhinitis independent

of eczema.23

It has been shown in patients suffering from severe eczema

that DCs, which are efficient antigen-presenting cells capable

of stimulating naıve antigen-specific T cells,33,34 are increased

in the epidermis.35 Wondering about the strong association

between IV and atopic diseases, we decided to study the pres-

ence of DC in the ichthyotic epidermis of patients with IV.

(In cases of concomitant eczema, biopsies were taken from

nonlesional skin exhibiting ichthyotic scaling but not eczema.)

CD1a is the main DC marker present on LC and IEDC.28 Inter-

estingly, atopic patients with a history of eczema as well

as atopic patients without eczema showed a significantly

increased rate of CD1a+ cells compared with nonatopic IV

skin (Fig. 5). The comparison of dendritic cell numbers of

atopic patients with IV with one mutation and those of atopic

patients harbouring two mutations showed no significant

difference (P > 0Æ6). In this context, it is of note that the

prevalence of atopic disorders in the IV subgroup with one

mutation did not differ from that in the subgroup with a

double mutation. However, it prompts us to speculate that the

increased CD1a+ cell numbers in IV patients reflect one of the

first steps of sensitization in IV. These observations lend fur-

ther support to the notion that there is a primary epidermal

barrier defect in IV that predisposes to atopic disorders. How-

ever, the results also demonstrate that filaggrin deficiency as

such does not necessarily lead to atopic manifestations and

that in addition, other pathogenic or genetic factors must be

involved.

Acknowledgments

We are grateful to all patients and control probands for partic-

ipation in this study and thank Tatjana Walker, Francis J.D.

Smith, Huijia Chen, Toshifumi Nomura, Peter Schulz, Stefan

Beissert, Wolfgang Kopcke, Peter Wissel, Jutta Buckmann and

Manfred Thomas for fruitful discussion and ⁄or excellent tech-

nical assistance. This work was supported by the Selbsthilfe

Ichthyose e.V., the Interdisciplinary Center of Clinical Research

University of Munster (Lo2 ⁄017 ⁄07), the GENESKIN European

coordination action and the Bundesministerium fur Bildung

und Forschung as part of the Network for rare diseases

NIRK (GFGM01143901). Filaggrin research in the McLean




laboratory is supported by grants from the British Skin Foun-

dation, The National Eczema Society, The Medical Research

Council (GO700314) and donations from anonymous families

affected by eczema in the Tayside Region of Scotland.

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Supporting information

Additional Supporting information may be found in the online

version of this article:

Fig S1. Overview of the sequence analysis of novel frameshift

mutations 424del17, 621del4 and 2974delGA. The first line

shows the sequences in heterozygous individuals, the second




line shows the ones for the wild type. In order to determine

the exact location of the frameshift mutations, sequence analy-

sis was performed with single strand DNA of the mutation

sequences, which were cloned into a pCR 2.1 vector. The

cloning sequence is given in the lower line. All frameshift

mutations (columns 1–3) result in preterminal stop codons:

after five codons for 424del17, after 15 codons for 621del4,

and after 34 codons for 2974delGA.

Fig S2. Overview of the established screening methods for the

novel mutations. 424del17, 621del4 and 2974delGA have

been screened by fragment analysis. R3766X and E4265X

were confirmed and screened by restriction enzyme analyses.

The fragment analysis of mutations indicated a heterozygous

genotype.

Table S1 Primer pairs used for the confirmation and screening

by restriction enzyme analysis.

Please note: Wiley-Blackwell are not responsible for the con-

tent or functionality of any supporting materials supplied by

the authors. Any queries (other than missing material) should

be directed to the corresponding author for the article.




ichthyosis vulgaris: novel flg mutations in the german population and high presence of cd1a+ cells...

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