- Email: [email protected]
Easy Method for Keratin 14 Gene Amplification to Exclude Pseudogene Sequences: New Keratin 5 and 14 Mutations in Epidermolysis Bullosa Simplex
LETTERS TO THE EDITOR
Easy Method for Keratin 14 Gene Amplification to Exclude Pseudogene 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 Epidermolysis bullosa simplex (EBS) is a group of hereditary mechanobullous disorders. Dominant negative mutations in the keratin 5 (KRT5, 12q13) and keratin 14 (KRT14, 17q12–q21) genes have been identified in most EBS patients (Fine et al., 1991; Irvine and McLean, 1999). In the most severe EBS subtype, Dowling-Meara (EBS-DM), a generalized herpetiform skin blistering is present. In contrast, the Ko¨bner form (EBS-K) shows a milder, generalized blistering, whereas blistering in EBS Weber-Cockayne (EBS-WC) typically involves the palms and soles (Sorensen et al., 2003). Molecular diagnosis of keratin disorders are complicated by the presence of dysfunctional pseudogenes (Smith, 2003). KRT14 has a truncated and a full-length inactive pseudogene (Savtchenko et al., 1988). To avoid KRT14 pseudogene amplification, additional methods have been established, such as long-range PCR and cDNA analyses (Wood et al., 2003), which are cost- and time-consuming methods. The most commonly applied genomic restriction digestion combined with PCR (Hut et al., 2000; Schuilenga-Hut et al., 2003) is often unreliable, because trace amounts of undigested sequences can be re-amplified, whereas overdigestion may result in noncompleted amplification due to star activity of the enzymes. The variable success of previously published methods led us to develop a new, single-step, allele-specific PCR to avoid sequence contamination from KRT14 pseudogenes. The KRT5 and
KRT14 mutations carry useful information about genotype–phenotype correlations. Evaluation of the new KRT14 mutation strategy: the new approach for KRT14 analysis proved to be a simply mutation analysis strategy in EBS that successfully excluded pseudogene sequence contamination. Two EBS- DM, one EBS-K, and six EBS-WC families were referred to us by DEBRA Hungary. They were unrelated and nonconsanguineous (Table 1). Study protocol, patient information sheet and patient consent forms were reviewed and approved by IRB (SE TUKEB 157-1997/98). Genomic DNA was isolated from 200 ml peripheral blood by NucleoSpin DNA kit (Macherey Nagel GmbH, Du¨ren, Germany). Exons 1–9 of KRT5 (AF274874.1, National Center for Biotechnology Information) and exons 1–8 of KRT14 (J00124, National Center for Biotechnology Information) were amplified. PCR was set up with 2 ImmoMix (Bioline, London, UK). Amplification conditions: hot-start initiation, 951C/ 10 minutes; 40 cycles, 951C/45 seconds; 30 seconds at optimal annealing temperature, 57–641C, 721C/30 seconds; final elongation, 721C/5 minutes. Annealing temperatures for KRT5 primers were described by Stephens et al. (1997) and Schuilenga-Hut et al. (2003). Considering the highly polymorphic nature of KRT14, we studied possible annealing sites for allele-specific primers, excluding sites with known intronic polymorphisms. Primers for KRT14 were designed by careful com-
Abbreviations: EBS, epidermolysis bullosa simplex; EBS-DM, epidermolysis bullosa simplex DowlingMeara; EBS-K, epidermolysis bullosa simplex Ko¨bner; EBS-WC, epidermolysis bullosa simplex WeberCockayne; KRT14, keratin 14 gene; KRT5, keratin 5 gene
& 2009 The Society for Investigative Dermatology
parison of the highly homologous full KRT14 pseudogene (NG_002781.1, National Center for Biotechnology Information) and the functional KRT14 (Table S1). Priming sites were chosen to bare nucleotide differences. Annealing sites were researched for intronic single nucleotide polymorphisms in genomic databases (Pubmed: GeneBank, Nucleotid/BLAST and single nucleotide polymorphism databases Altchul et al., (1997) (www.pubmed. com)), USSC Genome Bioinformatic database and BLAT (www.genome. ucsc.edu), ABi/CELERA single nucleotide polymorphism browser software (Applied Biosystems, Foster City, CA), single nucleotide polymorphism BLAST tool (www.snp.ims.u-tokyo.ac.jp). Primer specificity was reproofed with Primer3 (Rozen and Skaletsky, 2000) and VectorNTI (Invitrogen, Carlsbad, CA), annealing temperatures were determined by in silico predicament and confirmed by routine PCR runs. Amplicons generated with the newly designed primers were submitted to direct sequencing. Analysis by the restriction digestion method was also carried out in comparison. No pseudogene sequence contamination was detected by using the newly designed primers. Prescreening was routinely carried out with conformation-sensitive gel electrophoresis and heteroduplex analysis described earlier (Csiko´s et al., 2004, 2005). Exon 1 of both genes were not prescreened, but submitted to direct sequencing in all cases. Mutations and polymorphisms were reconfirmed by restriction digestion. If no restriction site was available, resequencing was repeatedly carried out. Allele frequencies were determined in the Hungarian population by 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 Pedigrees phenotype gene Exon domain cDNA
Triplet change
Mutation (amino-acid substitution) Validation
Allele 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
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
9
EBS-DM
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 samples (200 chromosomes; Table 1). We identified KRT14 mutations in four out of nine EBS families. The other five EBS pedigrees carried KRT5 mutations. Seven out of nine mutations share codons with those described previously in EBS (Table 1). All the identified mutations occur in the conserved codons of KRT5 or KRT14: none of the 200 chromosomes/100 studied controls carried the mutations. KRT5 mutations and genotype–phenotype correlations: in family 2 (EBSWC) the heterozygous c.547A4G, p.I183V was present. P.I183F, a different mutation in the same codon, had been previously described (Pfendner et al., 2003) in EBS-DM. Because of its smaller side chain, valin in P.I183V might cause less intraproteinic stress and steric distortion than phenylalanine in p.I183F, and this might explain the milder phenotype in our case. The affected members in family 3 (EBS-WC) carried the heterozygous mutation c.570G4C, p.E190D in exon 1. The previously reported p.E190K also induced a EBS-WC phenotype (Mu¨ller et al., 2006). 230
Family 5 (EBS-WC): in exon 5 a heterozygous substitution c.991C4G is present which results in p.R331G. Two other EBS-WC mutations at this codon (p.R331C, p.R331H) had been previously reported (Rugg et al., 1993; Mu¨ller et al., 2006). Families 1 (EBS-WC) and 4 (EBS-K) carried the known p.E170K and p.Q191P, respectively (Yasukawa et al., 2002; Mu¨ller et al., 2006), with the previously observed phenotype. KRT14 mutations and genotype–phenotype correlations: family 6 (EBS-WC) presented with the c.407T4A, p.L136Q mutation. Polarity change could lead to defected filament interactions and could be therefore pathogenic. In EBS-DM family 8, the affected proband carried the heterozygous c.1231G4A, p.E411K in exon 6. As the mutation was absent from the parents, a de novo mutation or gonadal mosaicism was probable. Known mutations of KRT14 at codon 411, p.E411X, Glu411del (c.1231_1233delGAG), have been associated with EBS-DM (Gu et al., 2002; Mu¨ller et al., 2006). All the affected members of family 9 (EBS-DM) were found to be heterozy-
Journal of Investigative Dermatology (2009), Volume 129
gous for a new T4A transversion c.1235T4A (exon 6), which resulted in the Ile4124Asn substitution (p.I412N). Family 7 (EBS-WC) carried the recurrent mutation p.R388C (Chen et al., 1995; Rugg et al., 2007) with the previously published phenotype. All the participating family members gave their written consent to mutation analysis. Protocol, patient information sheet and patient consent forms were reviewed and approved by IRB’s of the Semmelweis University and the Regional Ethincal Committee CentralHungary (SE TUKEB 157-14 1997/98). This study was conducted according to the Declaration of Helsinki principles. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We are grateful to our patients and their family members for their collaboration, as well as to the technical assistance of A´gnes Czippa´n, Katalin Barna, Marianna Ne´meth, Merce´desz Maza´n, and Ferencne´ Menyha´rt. This work was supported by ETT T-05-391/03, OTKA T043004, OTKA F049556, GENESKIN Coordination Action (LSHM-CT-2005-512117), Szenta´gothai Regional Knowledge Center and DebRA HUNGARY.
M Rostami-Yazdi et al. Pharmacokinetics of Fumaric Acid Esters
Annama´ria Gla´sz-Bo´na1,2, Ma´rta Medvecz1,2, Rachel Sajo´1, Re´ka Lepesi-Benko+ 1,2, Zsolt Tulassay2, Ma´ria Katona1, Zso´fia Hatvani1, Antal Blazsek1,2,3 and Sarolta Ka´rpa´ti1,2,3 1
Semmelweis University, Department of Dermatology, Venerologie and Dermatooncology, Budapest, Hungary; 2 Hungarian Academy of Sciences Semmelweis University Molecular Medicine Research Group, Budapest, Hungary and 3 Szenta´gothai Regional Knowledge Centre, Semmelweis University, Budapest, Hungary E-mail: [email protected]
SUPPLEMENTARY MATERIAL Table S1. Primers for KRT5 and KRT14 amplification.
REFERENCES Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, Miller W et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–402 Chen H, Bonifas JM, Matsumura K, Ikeda S, Leyden WA, Epstein EH Jr (1995) Keratin 14 gene mutations in patients with epidermolysis bullosa simplex. J Invest Dermatol 105: 629–32 Csiko´s M, Szalai Zs, Becker K, Sebo¨k B, Schneider I, Ka´rpa´ti S et al. (2004) Novel keratin 14 gene mutations in patients from Hungary with epidermolysis bullosa simplex. Exp Dermatol 13:185–91 Csiko´s M, Szo¨cs HI, La´szik A, Mecklenbeck S, Horva´th A, Ka´rpa´ti S et al. (2005) High frequency of the 425A–4G splice-site mutation and novel mutations of the COL7A1 gene in central Europe: significance for future mutation detection strategies in dystrophic
epidermolysis bullosa. Br J Dermatol 152: 879–86 Fine JD, Bauer EA, Briggaman RA, Carter DM, Eady RA, Esterly NB et al. (1991) Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J Am Acad Dermatol 24:119–35 Gu LH, Ichiki Y, Sato M, Kitajima Y novel nonsense mutation at E106 rod domain of keratin 14 causes epidermolysis bullosa simplex. J 29:136–45
(2002) A of the 2B dominant Dermatol
Hut PHL, v d Vlies P, Jonkman MF, Verlind E, Shimizu H, Buys CH et al. (2000) Exempting homologous pseudogene sequences from polymerase chain reaction amplification allows genomic keratin 14 hotspot mutation analysis. J Invest Dermatol 114:616–9 Irvine AD, McLean WH (1999) Human keratin disease: the increasing spectrum of disease and subtlety of the phenotype–genotype correlation. Br J Dermatol 140:815–28 Mu¨ller FB, Ku¨ster W, Wodecki K, Almeida H Jr, Bruckner-Tuderman L, Krieg T et al. (2006) Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa simplex: implications for disease phenotype and keratin filament assembly. Hum Mutat 27:719–20 Pfendner EG, Nakano A, Pulkkinen L, Christiano AM, Uitto J (2003) Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk. Prenat Diagn 23:447–56 Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386 Rugg EL, Horn HM, Smith FJ, Wilson NJ, Hill AJ, Magee GJ et al. (2007) Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations. J Invest Dermatol 127: 574–80
Rugg EL, Morley SM, Smith FJ, Boxer M, Tidman MJ, Navsaria H et al. (1993) Weber-Cockayne keratin mutations implicate the L12 linker domain in effective cytoskeleton function. Nat Genet 5:294–300 Savtchenko ES, Freedberg IM, Choi IY, Blumenberg M (1988) Inactivation of human keratin genes: the spectrum of mutations in the sequence of an acidic keratin pseudogene. Mol Biol Evol 5:97–108 Schuilenga-Hut PH, Vlies P, Jonkman MF, Waanders E, Buys CH, Scheffer H (2003) Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations. Hum Mutat 21:447–54 Smith F (2003) The molecular genetics of keratin disorders. Am J Clin Dermatol 5:347–64 Sorensen CB, Andresen BS, Jensen UB, Jensen TG, Jensen PK, Gregersen N et al. (2003) Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex. Exp Dermatol 12:472–9 Stephens K, Ehrlich P, Weaver M, Le R, Spencer A, Sybert VP (1997) Primers for exon-specific amplification of the KRT5 gene: identification of novel and recurrent mutations in epidermolysis bullosa simplex patients. J Invest Dermatol 108:349–53 Wood P, Baty DU, Lane EB, McLean WH (2003) Long-range polymerase chain reaction for specific full-length amplification of the human keratin 14 gene and novel keratin 14 mutations in epidermolysis bullosa simplex patients. J Invest Dermatol 120: 495–497 Yasukawa K, Sawamura D, McMillan JR, Nakamura H, Shimizu H (2002) Dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate filament assembly. J Biol 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 In the treatment of psoriasis, fumaric acid esters show good clinical efficacy combined with a favorable safety profile (Mrowietz et al., 1999).
Fumaderm, registered in Germany, consists of dimethylfumarate (DMF) and three salts of monoethylfumarate (MEF), and it has been shown that only DMF is required for clinical effect (Nieboer
Abbreviations: DMF, dimethylfumarate; GS-DMS, S-(1,2-dimethoxycarbonylethyl)glutathione; GSH, glutathione; MEF, monoethylfumarate; MMF, monomethylfumarate; NAC-DMS, N-acetyl-S-(1,2dimethoxycarbonylethyl)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(1-carboxy-2-methoxycarbonylethyl)cysteine and N-acetyl-S-(2-carboxy-1-methoxycarbonylethyl)-cysteine
et al., 1990). It is not yet clear whether DMF itself represents the active compound in vivo because only its hydrolysis product monomethylfumarate (MMF) could be detected in the plasma of healthy humans after oral intake (Litjens et al., 2004a). DMF exerts pharmacodynamic effects in low concentrations in vitro but could not be detected in vivo. In contrast, MMF showed in vitro effects only at concenwww.jidonline.org
231
Easy Method for Keratin 14 Gene Amplification to Exclude Pseudogene 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 Epidermolysis bullosa simplex (EBS) is a group of hereditary mechanobullous disorders. Dominant negative mutations in the keratin 5 (KRT5, 12q13) and keratin 14 (KRT14, 17q12–q21) genes have been identified in most EBS patients (Fine et al., 1991; Irvine and McLean, 1999). In the most severe EBS subtype, Dowling-Meara (EBS-DM), a generalized herpetiform skin blistering is present. In contrast, the Ko¨bner form (EBS-K) shows a milder, generalized blistering, whereas blistering in EBS Weber-Cockayne (EBS-WC) typically involves the palms and soles (Sorensen et al., 2003). Molecular diagnosis of keratin disorders are complicated by the presence of dysfunctional pseudogenes (Smith, 2003). KRT14 has a truncated and a full-length inactive pseudogene (Savtchenko et al., 1988). To avoid KRT14 pseudogene amplification, additional methods have been established, such as long-range PCR and cDNA analyses (Wood et al., 2003), which are cost- and time-consuming methods. The most commonly applied genomic restriction digestion combined with PCR (Hut et al., 2000; Schuilenga-Hut et al., 2003) is often unreliable, because trace amounts of undigested sequences can be re-amplified, whereas overdigestion may result in noncompleted amplification due to star activity of the enzymes. The variable success of previously published methods led us to develop a new, single-step, allele-specific PCR to avoid sequence contamination from KRT14 pseudogenes. The KRT5 and
KRT14 mutations carry useful information about genotype–phenotype correlations. Evaluation of the new KRT14 mutation strategy: the new approach for KRT14 analysis proved to be a simply mutation analysis strategy in EBS that successfully excluded pseudogene sequence contamination. Two EBS- DM, one EBS-K, and six EBS-WC families were referred to us by DEBRA Hungary. They were unrelated and nonconsanguineous (Table 1). Study protocol, patient information sheet and patient consent forms were reviewed and approved by IRB (SE TUKEB 157-1997/98). Genomic DNA was isolated from 200 ml peripheral blood by NucleoSpin DNA kit (Macherey Nagel GmbH, Du¨ren, Germany). Exons 1–9 of KRT5 (AF274874.1, National Center for Biotechnology Information) and exons 1–8 of KRT14 (J00124, National Center for Biotechnology Information) were amplified. PCR was set up with 2 ImmoMix (Bioline, London, UK). Amplification conditions: hot-start initiation, 951C/ 10 minutes; 40 cycles, 951C/45 seconds; 30 seconds at optimal annealing temperature, 57–641C, 721C/30 seconds; final elongation, 721C/5 minutes. Annealing temperatures for KRT5 primers were described by Stephens et al. (1997) and Schuilenga-Hut et al. (2003). Considering the highly polymorphic nature of KRT14, we studied possible annealing sites for allele-specific primers, excluding sites with known intronic polymorphisms. Primers for KRT14 were designed by careful com-
Abbreviations: EBS, epidermolysis bullosa simplex; EBS-DM, epidermolysis bullosa simplex DowlingMeara; EBS-K, epidermolysis bullosa simplex Ko¨bner; EBS-WC, epidermolysis bullosa simplex WeberCockayne; KRT14, keratin 14 gene; KRT5, keratin 5 gene
& 2009 The Society for Investigative Dermatology
parison of the highly homologous full KRT14 pseudogene (NG_002781.1, National Center for Biotechnology Information) and the functional KRT14 (Table S1). Priming sites were chosen to bare nucleotide differences. Annealing sites were researched for intronic single nucleotide polymorphisms in genomic databases (Pubmed: GeneBank, Nucleotid/BLAST and single nucleotide polymorphism databases Altchul et al., (1997) (www.pubmed. com)), USSC Genome Bioinformatic database and BLAT (www.genome. ucsc.edu), ABi/CELERA single nucleotide polymorphism browser software (Applied Biosystems, Foster City, CA), single nucleotide polymorphism BLAST tool (www.snp.ims.u-tokyo.ac.jp). Primer specificity was reproofed with Primer3 (Rozen and Skaletsky, 2000) and VectorNTI (Invitrogen, Carlsbad, CA), annealing temperatures were determined by in silico predicament and confirmed by routine PCR runs. Amplicons generated with the newly designed primers were submitted to direct sequencing. Analysis by the restriction digestion method was also carried out in comparison. No pseudogene sequence contamination was detected by using the newly designed primers. Prescreening was routinely carried out with conformation-sensitive gel electrophoresis and heteroduplex analysis described earlier (Csiko´s et al., 2004, 2005). Exon 1 of both genes were not prescreened, but submitted to direct sequencing in all cases. Mutations and polymorphisms were reconfirmed by restriction digestion. If no restriction site was available, resequencing was repeatedly carried out. Allele frequencies were determined in the Hungarian population by 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 Pedigrees phenotype gene Exon domain cDNA
Triplet change
Mutation (amino-acid substitution) Validation
Allele 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
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
9
EBS-DM
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 samples (200 chromosomes; Table 1). We identified KRT14 mutations in four out of nine EBS families. The other five EBS pedigrees carried KRT5 mutations. Seven out of nine mutations share codons with those described previously in EBS (Table 1). All the identified mutations occur in the conserved codons of KRT5 or KRT14: none of the 200 chromosomes/100 studied controls carried the mutations. KRT5 mutations and genotype–phenotype correlations: in family 2 (EBSWC) the heterozygous c.547A4G, p.I183V was present. P.I183F, a different mutation in the same codon, had been previously described (Pfendner et al., 2003) in EBS-DM. Because of its smaller side chain, valin in P.I183V might cause less intraproteinic stress and steric distortion than phenylalanine in p.I183F, and this might explain the milder phenotype in our case. The affected members in family 3 (EBS-WC) carried the heterozygous mutation c.570G4C, p.E190D in exon 1. The previously reported p.E190K also induced a EBS-WC phenotype (Mu¨ller et al., 2006). 230
Family 5 (EBS-WC): in exon 5 a heterozygous substitution c.991C4G is present which results in p.R331G. Two other EBS-WC mutations at this codon (p.R331C, p.R331H) had been previously reported (Rugg et al., 1993; Mu¨ller et al., 2006). Families 1 (EBS-WC) and 4 (EBS-K) carried the known p.E170K and p.Q191P, respectively (Yasukawa et al., 2002; Mu¨ller et al., 2006), with the previously observed phenotype. KRT14 mutations and genotype–phenotype correlations: family 6 (EBS-WC) presented with the c.407T4A, p.L136Q mutation. Polarity change could lead to defected filament interactions and could be therefore pathogenic. In EBS-DM family 8, the affected proband carried the heterozygous c.1231G4A, p.E411K in exon 6. As the mutation was absent from the parents, a de novo mutation or gonadal mosaicism was probable. Known mutations of KRT14 at codon 411, p.E411X, Glu411del (c.1231_1233delGAG), have been associated with EBS-DM (Gu et al., 2002; Mu¨ller et al., 2006). All the affected members of family 9 (EBS-DM) were found to be heterozy-
Journal of Investigative Dermatology (2009), Volume 129
gous for a new T4A transversion c.1235T4A (exon 6), which resulted in the Ile4124Asn substitution (p.I412N). Family 7 (EBS-WC) carried the recurrent mutation p.R388C (Chen et al., 1995; Rugg et al., 2007) with the previously published phenotype. All the participating family members gave their written consent to mutation analysis. Protocol, patient information sheet and patient consent forms were reviewed and approved by IRB’s of the Semmelweis University and the Regional Ethincal Committee CentralHungary (SE TUKEB 157-14 1997/98). This study was conducted according to the Declaration of Helsinki principles. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We are grateful to our patients and their family members for their collaboration, as well as to the technical assistance of A´gnes Czippa´n, Katalin Barna, Marianna Ne´meth, Merce´desz Maza´n, and Ferencne´ Menyha´rt. This work was supported by ETT T-05-391/03, OTKA T043004, OTKA F049556, GENESKIN Coordination Action (LSHM-CT-2005-512117), Szenta´gothai Regional Knowledge Center and DebRA HUNGARY.
M Rostami-Yazdi et al. Pharmacokinetics of Fumaric Acid Esters
Annama´ria Gla´sz-Bo´na1,2, Ma´rta Medvecz1,2, Rachel Sajo´1, Re´ka Lepesi-Benko+ 1,2, Zsolt Tulassay2, Ma´ria Katona1, Zso´fia Hatvani1, Antal Blazsek1,2,3 and Sarolta Ka´rpa´ti1,2,3 1
Semmelweis University, Department of Dermatology, Venerologie and Dermatooncology, Budapest, Hungary; 2 Hungarian Academy of Sciences Semmelweis University Molecular Medicine Research Group, Budapest, Hungary and 3 Szenta´gothai Regional Knowledge Centre, Semmelweis University, Budapest, Hungary E-mail: [email protected]
SUPPLEMENTARY MATERIAL Table S1. Primers for KRT5 and KRT14 amplification.
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epidermolysis bullosa. Br J Dermatol 152: 879–86 Fine JD, Bauer EA, Briggaman RA, Carter DM, Eady RA, Esterly NB et al. (1991) Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J Am Acad Dermatol 24:119–35 Gu LH, Ichiki Y, Sato M, Kitajima Y novel nonsense mutation at E106 rod domain of keratin 14 causes epidermolysis bullosa simplex. J 29:136–45
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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 In the treatment of psoriasis, fumaric acid esters show good clinical efficacy combined with a favorable safety profile (Mrowietz et al., 1999).
Fumaderm, registered in Germany, consists of dimethylfumarate (DMF) and three salts of monoethylfumarate (MEF), and it has been shown that only DMF is required for clinical effect (Nieboer
Abbreviations: DMF, dimethylfumarate; GS-DMS, S-(1,2-dimethoxycarbonylethyl)glutathione; GSH, glutathione; MEF, monoethylfumarate; MMF, monomethylfumarate; NAC-DMS, N-acetyl-S-(1,2dimethoxycarbonylethyl)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(1-carboxy-2-methoxycarbonylethyl)cysteine and N-acetyl-S-(2-carboxy-1-methoxycarbonylethyl)-cysteine
et al., 1990). It is not yet clear whether DMF itself represents the active compound in vivo because only its hydrolysis product monomethylfumarate (MMF) could be detected in the plasma of healthy humans after oral intake (Litjens et al., 2004a). DMF exerts pharmacodynamic effects in low concentrations in vitro but could not be detected in vivo. In contrast, MMF showed in vitro effects only at concenwww.jidonline.org
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