Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Letters to Dermatology or the existence of an epistatic relationship between Italian shared susceptibility haplotype (MIDDLE-ENDAL16) and FLG muta- tions. AD and PS patients selected either from trios analyzed in the previous work or newly recruited AD/PS patients were screened. A total of 195 PS patients and 178 AD patients were analyzed; 210 blood donors from the same ethnic background and without a history of PS, AD or other autoimmune disorders were selected as controls. All PS patients had a dermatologist-con- firmed diagnosis of chronic plaque psoriasis and most of them had type I psoriasis (70%). As for AD patients a consensus diag- nosis of the disease was assessed either by an expert dermatologist or by a pediatric allergologist. Written consent was obtained for all the patients or their parents. AD was extrinsic in 68% of the cases with 52% of the patients showing early onset of the disease ( !2 years old). Specific sensiti- zation was defined to be present if at least one of the specific IgE antibodies was positive (CAP-RAST class 61, corresponding to 60.35 kU/l). A raised total serum IgE was considered greater than 100 kU/l. Extrinsic AD was considered as AD with sensitization and/or IgE levels 6100 kU/l. The 2282del4 mutation was typed by sizing a fluorescently la- beled PCR fragment on an Applied Biosystems 3130xl as previ- ously described [7]. Genotyping of R501X was performed by TaqMan allelic discrimination assay (primer F: 5  -GCA CTG GAG GAA GAC AAG GAT-3  ; primer R: 5  -CTC TTG GGA CGC TGA ATG C; probe 1: 5  -CTG TCT CGT GCC TGC-3  ; probe 2: 5  -CTG TCT CAT GCC TGC-3 ). Genotype assessment of both 2282del4 and R501X was confirmed by direct sequencing of 10% of the samples. Screening of FLG mutations was performed as previously described [5]. A PCR fragment of 3697 bp was am- plified from human genomic DNA using forward primer FILF3 (5  -GCT GAT AAT GTG ATT CTG TCT G-3 ) and reverse prim- er RPT3P10R (5  -GAC CCC GAT GAT TGT TCC TGT-3 ). This PCR fragment encompasses the 5  -end of exon 3 of the pro- filaggrin gene, repeats 1 and 2 and 401 bp of repeat 3. PCR condi- tions were as follows: (94 ° C 5 min) 1, (94 ° C 30 s, 61 ° C 45 s, 72 ° C 3 min 20 s) 31 cycles, and a final extension at 72 ° C for 5 min. The 3,697-bp PCR fragment was sequenced with primers FILr3.F1 (5  -GGG TCA GGA CAC CAT TCG TGC-3 ) and RPT3P10R. Genotyping of FLG mutations in PS patients revealed that the frequency of risk alleles of R501X and 2282del4 was 0.0%, i.e. no mutant allele has been observed (table 1). Genotyping of R501X and 2282del4 mutations in AD patients and controls showed sim- ilar frequency of risk alleles: 0.6 vs. 0.0% and 0.9 vs. 0.5%, respec- tively. Risk allele frequencies observed in the Italian AD patients were strongly reduced in respect to those described in other pa- tients of European origin [4, 6]. Following the above-mentioned results we searched for additional mutations in FLG. We rese- quenced 120 AD patients and 100 PS patients for 5  -end of exon 3 of the profilaggrin gene, repeats 1 and 2 and 401 bp of repeat 3. We did not observe additional mutations in FLG in either AD or PS patients. These results taken together with very recent data [8] seem to rule out an involvement of R501X and 2282del4 in PS and Key Words Psoriasis  Atopic dermatitis  Filaggrin  Complex disease Psoriasis (PS) is a chronic inflammatory skin disorder charac- terized by keratinocyte hyperproliferation and altered differen- tiation. Atopic dermatitis (AD) is a common chronic inflamma- tory skin disease characterized by itchy inflamed skin. Genome- wide linkage studies for PS/AD revealed a significant linkage to a region on chromosome 1q21 [1, 2], containing the epidermal dif- ferentiation complex (EDC). EDC is a cluster of genes with a key role in terminal differentiation of human epidermis. Recently we demonstrated a co-localization of the PS (PSORS4) and AD (ATOD2) susceptibility loci in a 42-kb interval in Italian patients [3]. Most recently, it has been reported that two loss-of-function mutations – R501X and 2282del4 – within the filaggrin gene ( FLG) located in the epidermal differentiation complex are associ- ated with AD in several populations [4–6]. Loss-of-function mu- tations in FLG predispose to ichthyosis [7], which in its mild form shares clinical characteristics with PS and AD such as dryness and altered differentiation of keratinocytes. In the outer granular layer of the epidermis, filaggrin is associated with keratin inter- mediate filaments, supports their packing into bundles and plays a critical role in carrying out the protective task of epidermis. In terminally differentiated keratinocytes, filaggrin is cross-linked to the cornified cell envelope, which constitutes an insoluble bar- rier of the stratum corneum protecting the organism against en- vironmental agents and preventing epidermal water loss. The loss of epidermal barrier function represents one of the first steps in the development of PS and AD and therefore FLG can be consid- ered an obvious predisposing gene to both diseases. Although FLG is located 1 Mb from the susceptibility haplo- type associated to both PS and AD in Italian patients [3], FLG null mutations were genotyped in a PS and AD cohort in order to eval- uate either their involvement in the pathogenesis of both diseases © 2008 S. Karger AG, Basel Accessible online at: www.karger.com/drm Dermatology 200 ;216:83–84 DOI: 10.1159/000109365 R501X and 2282del4 Filaggrin Mutations Do Not Confer Susceptibility to Psoriasis and Atopic Dermatitis in Italian Patients Emiliano Giardina a , Nicoletta Paolillo a , Cecilia Sinibaldi a , Giuseppe Novelli a, b a Department of Biopathology and Centre of Excellence for Genomic risk Assessment in Multifactorial and Complex Diseases, School of Medicine, University of Rome ‘Tor Vergata’, Rome, Italy; b Department of Cardiovascular Medicine, University of Arkansas for Medical Sciences, Little Rock, Ark., USA 8