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Easy Method for Keratin 14 Amplification to Exclude Sequences: New Keratin 5 and 14 Mutations in Epidermolysis Bullosa Simplex

Journal of Investigative Dermatology (2009) 129, 229–231; doi:10.1038/jid.2008.223; published online 14 August 2008

TO THE EDITOR KRT14 mutations carry useful informa- parison of the highly homologous full Epidermolysis bullosa simplex (EBS) is a tion about genotype–phenotype corre- KRT14 pseudogene (NG_002781.1, group of hereditary mechanobullous lations. National Center for Biotechnology In- disorders. Dominant negative muta- Evaluation of the new KRT14 muta- formation) and the functional KRT14 tions in the keratin 5 (KRT5, 12q13) tion strategy: the new approach for (Table S1). Priming sites were chosen to and keratin 14 (KRT14, 17q12–q21) KRT14 analysis proved to be a simply bare nucleotide differences. have been identified in most mutation analysis strategy in EBS that Annealing sites were researched for EBS patients (Fine et al., 1991; Irvine successfully excluded pseudogene se- intronic single nucleotide polymorph- and McLean, 1999). In the most severe quence contamination. isms in genomic databases (Pubmed: EBS subtype, Dowling-Meara (EBS-DM), Two EBS- DM, one EBS-K, and six GeneBank, Nucleotid/BLAST and single a generalized herpetiform skin blister- EBS-WC families were referred to us by nucleotide polymorphism databases ing is present. In contrast, the Ko¨bner DEBRA Hungary. They were unrelated Altchul et al., (1997) (www.pubmed. form (EBS-K) shows a milder, general- and nonconsanguineous (Table 1). com)), USSC Genome Bioinformatic ized blistering, whereas blistering in Study protocol, patient information database and BLAT (www.genome. EBS Weber-Cockayne (EBS-WC) typically sheet and patient consent forms were ucsc.edu), ABi/CELERA single nucleo- involves the palms and soles (Sorensen reviewed and approved by IRB (SE tide polymorphism browser software et al., 2003). TUKEB 157-1997/98). (Applied Biosystems, Foster City, CA), Molecular diagnosis of keratin dis- Genomic DNA was isolated from single nucleotide polymorphism BLAST orders are complicated by the presence 200 ml peripheral blood by NucleoSpin tool (www.snp.ims.u-tokyo.ac.jp). Primer of dysfunctional (Smith, DNA kit (Macherey Nagel GmbH, specificity was reproofed with Primer3 2003). KRT14 has a truncated and a Du¨ren, Germany). (Rozen and Skaletsky, 2000) and Vec- full-length inactive pseudogene Exons 1–9 of KRT5 (AF274874.1, torNTI (Invitrogen, Carlsbad, CA), anneal- (Savtchenko et al., 1988). To avoid National Center for Biotechnology In- ing temperatures were determined by in KRT14 pseudogene amplification, addi- formation) and exons 1–8 of KRT14 silico predicament and confirmed by tional methods have been established, (J00124, National Center for Biotech- routine PCR runs. such as long-range PCR and cDNA nology Information) were amplified. generated with the newly analyses (Wood et al., 2003), which PCR was set up with 2 ImmoMix designed primers were submitted to are cost- and time-consuming methods. (Bioline, London, UK). Amplification direct sequencing. Analysis by the The most commonly applied genomic conditions: hot-start initiation, 951C/ restriction digestion method was also restriction digestion combined with 10 minutes; 40 cycles, 951C/45 sec- carried out in comparison. No pseudo- PCR (Hut et al., 2000; Schuilenga-Hut onds; 30 seconds at optimal annealing gene sequence contamination was et al., 2003) is often unreliable, because temperature, 57–641C, 721C/30 sec- detected by using the newly designed trace amounts of undigested sequences onds; final elongation, 721C/5 minutes. primers. can be re-amplified, whereas overdi- Annealing temperatures for KRT5 pri- Prescreening was routinely carried gestion may result in noncompleted mers were described by Stephens et al. out with conformation-sensitive gel amplification due to star activity of the (1997) and Schuilenga-Hut et al.(2003). electrophoresis and heteroduplex enzymes. Considering the highly polymorphic analysis described earlier (Csiko´s The variable success of previously nature of KRT14, we studied possible et al., 2004, 2005). Exon 1 of both published methods led us to develop a annealing sites for allele-specific pri- genes were not prescreened, but sub- new, single-step, allele-specific PCR to mers, excluding sites with known in- mitted to direct sequencing in all cases. avoid sequence contamination from tronic polymorphisms. Primers for Mutations and polymorphisms were KRT14 pseudogenes. The KRT5 and KRT14 were designed by careful com- reconfirmed by restriction digestion. If no restriction site was available, resequencing was repeatedly carried Abbreviations: EBS, epidermolysis bullosa simplex; EBS-DM, epidermolysis bullosa simplex Dowling- Meara; EBS-K, epidermolysis bullosa simplex Ko¨bner; EBS-WC, epidermolysis bullosa simplex Weber- out. Allele frequencies were deter- Cockayne; KRT14, keratin 14 gene; KRT5, keratin 5 gene mined in the Hungarian population by

& 2009 The Society for Investigative Dermatology www.jidonline.org 229 A Gla´sz-Bo´na et al. Mutation analysis of KRT5 and KRT14 genes

Table 1. KRT5 and KRT14 mutations and polymorphisms in EBS families Affected Clinical Keratin keratin Triplet Mutation Allele Pedigrees phenotype gene Exon domain cDNA change (amino-acid substitution) Validation Inheritance frequency

1 EBS-WC KRT5 1 1A c.508G4A GAG-AAG p.E170K (Glu-Lys) Sequencing Paternal — 2 EBS-WC KRT5 1 1A c.547A4G ATC-GTC p.I183V (Ile-Val) Sequencing Paternal — KRT 5 1 1A c.382G4C GGT-CGT p.G128R (Gly-Arg) Sequencing Paternal 0.59 3 EBS-WC KRT5 2 1A c.570G4C GAG-GAC p.E190D (Glu-Asp) TseI. (–) Paternal — KRT5 1 1B c.513G4A CAG-GAA p.Q171Q (Gln-Gln) Sequencing Maternal 0.54 4 EBS-K KRT5 2 1A c.572A4C CAG-CCG p.Q191P (Gln-Pro) TseI. (–) Paternal — 5 EBS-WC KRT5 5 L1-2 c.991C4G CGC-GGC p.R331G (Arg-Gly) AciI. (–) Paternal — KRT5 1 Head c.156C4A GCC-GCA p.A52A (Ala-Ala) Sequencing Paternal 0.34 6 EBS-WC KRT14 1 1A c.407T4A CTG-CAG p.L136Q (Leu-Gln) DdeI. (+) Maternal — 7 EBS-WC KRT14 6 2B c.1162C4T CGC-TGC p.R388C (Arg-Cys) AciI. (–) Paternal — 8 EBS-DM KRT14 6 2B c.1231G4A GAG-AAG p.E411K (Glu-Lys) MboII. (+) De Novo — KRT5 1 1B c.630T4C ACC-ACA p.T210T (Thr-Thr) Sequencing Maternal 0.66 9 EBS-DM KRT14 6 2B c.1235T4A ATC-AAC p.I412N (Ile-Asn) Sau3AI. (+) Paternal — KRT14 1 Head c.369T4C AAT-AAC p.N123N (Asn-Asn) Sequencing Maternal 0.38 EBS-DM, epidermolysis bullosa simplex Dowling-Meara; EBS-K, epidermolysis bullosa simplex Ko¨bner; EBS-WC, epidermolysis bullosa simplex Weber-Cockayne. Novel mutations are indicated in bold, polymorphisms in italic letters; () mutation abolishes the restriction site; (+) mutation generates the restriction site. Allele frequencies are calculated upon studies in 100 unrelated control individuals (200 chromosomes).

screening 100 non-EBS control DNA Family 5 (EBS-WC): in exon 5 a gous for a new T4A transversion samples (200 chromosomes; Table 1). heterozygous substitution c.991C4Gis c.1235T4A (exon 6), which resulted We identified KRT14 mutations in present which results in p.R331G. Two in the Ile4124Asn substitution four out of nine EBS families. The other other EBS-WC mutations at this codon (p.I412N). five EBS pedigrees carried KRT5 muta- (p.R331C, p.R331H) had been pre- Family 7 (EBS-WC) carried the re- tions. Seven out of nine mutations share viously reported (Rugg et al., 1993; current mutation p.R388C (Chen et al., codons with those described previously Mu¨ller et al., 2006). 1995; Rugg et al., 2007) with the in EBS (Table 1). All the identified Families 1 (EBS-WC) and 4 (EBS-K) previously published phenotype. mutations occur in the conserved co- carried the known p.E170K and All the participating family members dons of KRT5 or KRT14: none of the p.Q191P, respectively (Yasukawa gave their written consent to mutation 200 chromosomes/100 studied controls et al., 2002; Mu¨ller et al., 2006), with analysis. Protocol, patient information carried the mutations. the previously observed phenotype. sheet and patient consent forms were KRT5 mutations and genotype–phe- KRT14 mutations and genotype–phe- reviewed and approved by IRB’s of the notype correlations: in family 2 (EBS- notype correlations: family 6 (EBS-WC) Semmelweis University and the WC) the heterozygous c.547A4G, presented with the c.407T4A, Regional Ethincal Committee Central- p.I183V was present. P.I183F, a differ- p.L136Q mutation. Polarity change Hungary (SE TUKEB 157-14 1997/98). ent mutation in the same codon, had could lead to defected filament interac- This study was conducted according to been previously described (Pfendner tions and could be therefore pathogenic. the Declaration of Helsinki principles. et al., 2003) in EBS-DM. Because of In EBS-DM family 8, the affected its smaller side chain, valin in P.I183V proband carried the heterozygous CONFLICT OF INTEREST might cause less intraproteinic stress c.1231G4A, p.E411K in exon 6. As The authors state no conflict of interest. and steric distortion than phenylalanine the mutation was absent from the in p.I183F, and this might explain the parents, a de novo mutation or gonadal ACKNOWLEDGMENTS milder phenotype in our case. mosaicism was probable. Known muta- We are grateful to our patients and their family members for their collaboration, as well as to the The affected members in family 3 tions of KRT14 at codon 411, p.E411X, technical assistance of A´ gnes Czippa´n, Katalin (EBS-WC) carried the heterozygous Glu411del (c.1231_1233delGAG), Barna, Marianna Ne´meth, Merce´desz Maza´n, and mutation c.570G4C, p.E190D in exon have been associated with EBS-DM Ferencne´ Menyha´rt. This work was supported by 1. The previously reported p.E190K also (Gu et al., 2002; Mu¨ller et al., 2006). ETT T-05-391/03, OTKA T043004, OTKA F049556, GENESKIN Coordination Action induced a EBS-WC phenotype (Mu¨ller All the affected members of family 9 (LSHM-CT-2005-512117), Szenta´gothai Regional et al., 2006). (EBS-DM) were found to be heterozy- Knowledge Center and DebRA HUNGARY.

230 Journal of Investigative Dermatology (2009), Volume 129 M Rostami-Yazdi et al. Pharmacokinetics of Fumaric Acid Esters

Annama´ria Gla´sz-Bo´na1,2,Ma´rta epidermolysis bullosa. Br J Dermatol 152: Rugg EL, Morley SM, Smith FJ, Boxer M, Tidman 1,2 ´ 1 ´ 879–86 MJ, Navsaria H et al. (1993) Weber-Cock- Medvecz , Rachel Sajo ,Reka ayne keratin mutations implicate the L12 +1,2 2 Fine JD, Bauer EA, Briggaman RA, Carter DM, Lepesi-Benko , Zsolt Tulassay ,Ma´ria linker domain in effective cytoskeleton func- Eady RA, Esterly NB et al. (1991) Revised 1 1 tion. Nat Genet 5:294–300 Katona , Zso´fia Hatvani , Antal clinical and laboratory criteria for subtypes of 1,2,3 1,2,3 Blazsek and Sarolta Ka´rpa´ti inherited epidermolysis bullosa. J Am Acad Savtchenko ES, Freedberg IM, Choi IY, Blumen- berg M (1988) Inactivation of human keratin 1Semmelweis University, Department of Dermatol 24:119–35 genes: the spectrum of mutations in the Dermatology, Venerologie and Gu LH, Ichiki Y, Sato M, Kitajima Y (2002) A sequence of an acidic keratin pseudogene. Dermatooncology, Budapest, Hungary; novel nonsense mutation at E106 of the 2B Mol Biol Evol 5:97–108 2Hungarian Academy of Sciences - rod domain of keratin 14 causes dominant Semmelweis University Molecular Medicine epidermolysis bullosa simplex. J Dermatol Schuilenga-Hut PH, Vlies P, Jonkman MF, Waan- ders E, Buys CH, Scheffer H (2003) Mutation Research Group, Budapest, Hungary and 29:136–45 analysis of the entire keratin 5 and 14 genes 3Szenta´gothai Regional Knowledge Centre, Hut PHL, v d Vlies P, Jonkman MF, Verlind E, in patients with epidermolysis bullosa sim- Semmelweis University, Budapest, Hungary Shimizu H, Buys CH et al. (2000) Exempting plex and identification of novel mutations. E-mail: [email protected] homologous pseudogene sequences from Hum Mutat 21:447–54 polymerase chain reaction amplification al- Smith F (2003) The molecular of lows genomic keratin 14 hotspot mutation SUPPLEMENTARY MATERIAL keratin disorders. Am J Clin Dermatol analysis. J Invest Dermatol 114:616–9 5:347–64 Table S1. Primers for KRT5 and KRT14 amplifica- Irvine AD, McLean WH (1999) Human keratin Sorensen CB, Andresen BS, Jensen UB, Jensen TG, tion. disease: the increasing spectrum of disease Jensen PK, Gregersen N et al. (2003) Func- and subtlety of the phenotype–genotype tional testing of keratin 14 mutant proteins REFERENCES correlation. Br J Dermatol 140:815–28 associated with the three major subtypes of Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Mu¨ller FB, Ku¨ster W, Wodecki K, Almeida H Jr, epidermolysis bullosa simplex. Exp Dermatol Zhang Z, Miller W et al. (1997) Gapped Bruckner-Tuderman L, Krieg T et al. (2006) 12:472–9 BLAST and PSI-BLAST: a new generation of Novel and recurrent mutations in keratin Stephens K, Ehrlich P, Weaver M, Le R, Spencer A, protein database search programs. Nucleic KRT5 and KRT14 genes in epidermolysis Sybert VP (1997) Primers for exon-specific Acids Res 25:3389–402 bullosa simplex: implications for disease amplification of the KRT5 gene: identification of novel and recurrent mutations in epider- Chen H, Bonifas JM, Matsumura K, Ikeda S, phenotype and keratin filament assembly. molysis bullosa simplex patients. J Invest Leyden WA, Epstein EH Jr (1995) Keratin 14 Hum Mutat 27:719–20 Dermatol 108:349–53 gene mutations in patients with epidermoly- Pfendner EG, Nakano A, Pulkkinen L, Christiano sis bullosa simplex. J Invest Dermatol 105: AM, Uitto J (2003) Prenatal diagnosis for Wood P, Baty DU, Lane EB, McLean WH (2003) 629–32 epidermolysis bullosa: a study of 144 con- Long-range polymerase chain reaction for specific full-length amplification of the Csiko´s M, Szalai Zs, Becker K, Sebo¨kB, secutive pregnancies at risk. Prenat Diagn 23:447–56 human keratin 14 gene and novel keratin Schneider I, Ka´rpa´ti S et al. (2004) Novel 14 mutations in epidermolysis bullosa keratin 14 gene mutations in patients from Rozen S, Skaletsky H (2000) Primer3 on the simplex patients. J Invest Dermatol 120: Hungary with epidermolysis bullosa simplex. WWW for general users and for biologist 495–497 Exp Dermatol 13:185–91 programmers. Methods Mol Biol 132: Yasukawa K, Sawamura D, McMillan JR, 365–386 Csiko´s M, Szo¨cs HI, La´szik A, Mecklenbeck S, Nakamura H, Shimizu H (2002) Dominant Horva´th A, Ka´rpa´ti S et al. (2005) High Rugg EL, Horn HM, Smith FJ, Wilson NJ, Hill AJ, and recessive compound heterozygous muta- frequency of the 425A–4G splice-site muta- Magee GJ et al. (2007) Epidermolysis bullosa tions in epidermolysis bullosa simplex de- tion and novel mutations of the COL7A1 simplex in Scotland caused by a spectrum of monstrate the role of the stutter region in gene in central Europe: significance for future keratin mutations. J Invest Dermatol 127: keratin intermediate filament assembly. J Biol mutation detection strategies in dystrophic 574–80 Chem 277:23670–4

Detection of Metabolites of Fumaric Acid Esters in Human Urine: Implications for Their Mode of Action

Journal of Investigative Dermatology (2009) 129, 231–234; doi:10.1038/jid.2008.197; published online 14 August 2008

TO THE EDITOR Fumaderm, registered in Germany, et al., 1990). It is not yet clear whether In the treatment of psoriasis, fumaric consists of dimethylfumarate (DMF) and DMF itself represents the active com- acid esters show good clinical efficacy three salts of monoethylfumarate (MEF), pound in vivo because only its hydro- combined with a favorable safety pro- and it has been shown that only DMF is lysis product monomethylfumarate file (Mrowietz et al., 1999). required for clinical effect (Nieboer (MMF) could be detected in the plasma of healthy humans after oral intake (Litjens et al., 2004a). Abbreviations: DMF, dimethylfumarate; GS-DMS, S-(1,2-dimethoxycarbonylethyl)glutathione; DMF exerts pharmacodynamic effects GSH, glutathione; MEF, monoethylfumarate; MMF, monomethylfumarate; NAC-DMS, N-acetyl-S-(1,2- in low concentrations in vitro but could dimethoxycarbonylethyl)cysteine; NAC-MES, mixture of N-acetyl-S-(1-carboxy-2-ethoxycarbonylethyl)- cysteine and N-acetyl-S-(2-carboxy-1-ethoxycarbonylethyl)cysteine; NAC-MMS, mixture of N-acetyl-S- not be detected in vivo. In contrast, MMF (1-carboxy-2-methoxycarbonylethyl)cysteine and N-acetyl-S-(2-carboxy-1-methoxycarbonylethyl)-cysteine showed in vitro effects only at concen-

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