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Two Novel Mutations in the Keratin 1 Gene in Epidermolytic Hyperkeratosis
LETTER TO THE EDITOR
Two Novel Mutations in the Keratin 1 Gene in Epidermolytic Hyperkeratosis the KRT2e gene was reported in ichthyosis bullosa of Siemens (Whittock et al, 2001). In patient EHK-AS, we found a single nucleotide substitution on one allele of codon 475 in exon 7 of the KRT1 gene (CTG to CCG) so that the sequence gel of Fig 1(b) shows both a wild-type T and a mutant C at nucleotide position 1457 (GenBank NM-006121; relative to the translation start point) in the proband. The mutant allele encodes a proline residue instead of a wild-type leucine residue in position 113 of 2B rod domain segment. This nucleotide substitution destroys an MspI restriction enzyme site (C/CGG), which was used to con®rm that the unaffected parents and 50 other unrelated unaffected persons do not posses this new mutation.
To the Editor: Epidermolytic hyperkeratosis (EHK, MIM113800) is a rare autosomal dominant genodermatosis, although many cases occur sporadically as new mutations. It is characterized by blistering and erythema at birth, and development of hyperkeratosis with increasing age. Ultrastructural analyses reveal clumping of the keratin intermediate ®laments (KIF) within suprabasal keratinocytes of the epidermis. Previous studies have demonstrated that EHK is caused by mutations in the genes encoding the keratin 1 (K1) or the K10 proteins (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992; McLean and Lane, 1995; Korge and Krieg, 1996). Clinically, EHK has been divided into the palmoplantar type (palm sole, PS) and the nonpalmoplantar type (nonpalm sole), and each type has been subdivided into three subtypes according to the nature and severity of scaling (DiGiovanna and Bale, 1994; DiGiovanna et al, 1998). The mutation/clinical phenotype correlations available suggest that the PS and nonpalm sole types are due primarily to mutations in the KRT1 and KRT10 genes, respectively. The more severe PS-1 and PS-2 cases are caused by mutations that change sequences encoding the beginning of the 1A rod domain segment, or towards the end of the 2B rod domain segment (Yang et al, 1994, 1999; Arin et al, 1999, 2000; Cserhalmi-Friedman et al, 2000). In this study, we describe two separate cases of EHK, classi®ed as PS-2, caused by two different novel mutations: Asn187Lys located in exon 1, which is the 8th residue of the 1A rod domain segment; and Leu475Pro located in exon 7, which is the 10th residue from the end of 2B rod domain segment of K1. Two unrelated children, EHK-SJ and EHK-AS, have diffuse thick keratoderma on both palms and soles as well as moderate scaly patches on their entire bodies, but the morphology of their hair, nails, and teeth are normal. Light microscopic examination revealed vacuolar degeneration in the spinous and granular layers. As both sets of parents were normal, these represent sporadic cases. Both were classi®ed as having the PS-2 form of EHK. DNA was extracted (Chipev et al, 1994; Yang et al, 1994) from both the affected and unaffected family members, and from 50 unrelated normal individuals used as controls. Following sequencing of all exons of the KRT1 and KRT10 genes, patient EHK-SJ had a single nucleotide substitution on one allele of codon 187 in exon 1 of the KRT1 gene (AAC to AAA) so that the sequence gel of Fig 1(a) shows both the wild-type C and a mutant A at nucleotide position 564 (GenBank NM-006121; relative to the translation start point) in the proband. The mutant allele encodes a lysine residue instead of a wild-type asparagine residue in position 8 of the 1A rod domain segment. As this nucleotide substitution does not create or destroy a restriction enzyme site, we performed a polymerase chain reaction ampli®cation of a speci®c allele assay (Yang et al, 1994, 1999). As the parents and 50 other unrelated normal controls do not have this substitution, it represents a new mutation. The same mutation in Manuscript received June 7, 2002; accepted for publication June 10, 2002 Reprint requests to: June-Mo Yang, MD, PhD, Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, 50 Il-won Dong, Kangnam Ku, Seoul, 135-710, Republic of Korea. Email: [email protected] 0022-202X/02/$15.00
Figure 1. (a) EHK-SJ. DNA sequences showing C to A change (arrow), which introduces an Asn187Lys amino acid substitution in the 1A rod domain segment of K1. (b)EHK-AS. DNA sequences showing T to C change (arrow), which introduces a Leu475Pro amino acid substitution in the 2B rod domain segment of K1.
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 976
VOL. 119, NO. 4 OCTOBER 2002
LETTERS TO THE EDITOR
977
Next, we synthesized four 18-residue peptides corresponding to the beginning of the 1A and the end of 2B region of wild-type K1, or sequences with the described amino acid substitutions, respectively. These were used for an assay that uses synthetic peptides to interfere with KIF assembly (Chipev et al, 1992): wild-type peptides disassemble KIF, whereas peptides bearing identi®ed substitutions caused by novel mutations often do not. In both cases, we found that the wild-type 1A or 2B peptides interfered with the formation of KIF in vitro; however, neither mutant peptide interfered with KIF assembly (Fig 2). From this we can conclude that the substitutions resulting from the observed mutations affect normal KIF structure. The three-dimensional structures of the 1A (Strelkov et al, 2002) and 2B (Herrmann et al, 2000) domains of the related IF protein vimentin have been solved. As the amino acid sequences of vimentin and K1 are conserved in these regions, their structures are likely to be similar. An asparagine residue in position 8 of the 1A domain has been conserved in all IF proteins. Its side chain projects laterally outward from the axis of the coiled-coil, where it presumably interacts with residues on an adjacent molecule. Indeed, we have predicted that this residue forms H-bonds with residues in the L2 linker region in the A11 alignment mode of two adjacent molecules in the tetramer (Mehrani et al, 2001). By model building analyses, the substitution of a larger positively charged lysine residue will interfere with such interactions. Thus this Asn187Lys substitution in patient EHK-SJ will likely severely interfere with KIF structure at the twomolecule hierarchical stage of KIF assembly. Likewise, a leucine residue in position 113 of the 2B rod domain segment has been conserved in all IF chains. Based on the extant model of this region in vimentin, this leucine residue marks the point at which the coiledcoil 2B sequences unravel and fold back along the axis of the coiledcoil. Insertion of an aberrant cis peptide bond of a proline residue at this point will prevent this refold. Accordingly, the Leu475Pro substitution will cause failed KIF assembly at the two-molecule level of assembly. We thank the families for their participation and cooperation, and Dr U. Aebi for sharing the structure of vimentin before publication. This work was supported by a grant (09-PJ3-PG6-01GN12-0001) of the 2001 Good Health R & D Project, Ministry of Health and Welfare, Republic of Korea (Yang).
Dong-Youn Lee,*² Kwang-Sung Ahn,² Cha-Hui Lee,² NarkKyoung Rho,* Joo-Heung Lee,* Eil-Soo Lee,* Peter M. Steinert,³ and Jun-Mo Yang*² *Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, ²Clinical Research Center, Samsung Biomedical Research Institute, Seoul, Republic of Korea, ³Laboratory of Skin Biology, National Institutes of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, U.S.A. REFERENCES Arin MJ, Longley MA, Kuster W, Huber M, Hohl D, Rothnagel JA, Roop DR: An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis. Exp Dermatol 8:124±127, 1999 Arin MJ, Longley MA, Epstein EH, Rothnagel JA, Roop DR: Identi®cation of a novel mutation in keratin 1 in a family with epidermolytic hyperkeratosis. Exp Dermatol 9:16±19, 2000 Cheng J, Snyder AJ, Yu Q-C, Paller AS, Fuchs E: The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-speci®c genes. Cell 70:811±819, 1992 Chipev CC, Korge BP, Markova N, Bale SJ, DiGiovanna JJ, Compton JG, Steinert PM: A leucine-proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 70:821±828, 1992 Chipev CC, Yang J-M, DiGiovanna JJ, Steinert PM, Marekov LN, Compton JG, Bale SJ: Preferential sites in keratin 10 that are mutated in epidermolytic hyperkeratosis. Am J Hum Genet 54:179±190, 1994 Cserhalmi-Friedman PB, Squeo R, Gordon D, Garzon M, Schneiderman P, Grossman ME, Christiano AM: Epidermolyic hyperkeratosis in a Hispanic family resulting from a mutation in the keratin 1 gene. Clin Exp Dermatol 25:241±243, 2000
Figure 2. KIF disassembly assay using 18-residue wild-type 1A (b) or 2B (d) peptides. In both cases, normal KIF (a) are disassembled into small particles within 30 min at 23°; however, the Asn187Lys mutant 1A peptide (c) or Leu475Pro mutant 2B peptide (e) had no discernible effect on preformed KIF, or KIF assembly in vitro (not shown). The assay suggests that the structure of the mutant portions of the proteins will interfere with KIF assembly. Scale bar = 200 nm. DiGiovanna JJ, Bale SJ: Clinical heterogeneity in epidermoltyic hyperkeratosis. Arch Dermatol 130:1026±1035, 1994 DiGiovanna JJ, Bale SJ, Steinert PM: Epidermolytic hyperkeratosis. In: Kupper T (ed.). Textbook of Molecular Medicine, Part 8, Molecular Mechanisms in Skin and Connective Tissues. Totowa: Human Press, 1998, 707±712 Herrmann H, Strelkov S, Feja B, et al: The intermediate ®lament protein consensus motif of helix 2B: atomic structure and contribution to assembly. J Mol Biol 298:817±832, 2000 Korge BP, Krieg T: The molecular basis for inherited bullous diseases. J Mol Med 74:59±70, 1996 McLean WHI, Lane EB: Intermediate ®laments in disease. Curr Opin Cell Biol 7:118± 125, 1995 Mehrani T, Morasso MI, Marekov LN, Parry DAD, Steinert PM: Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate ®laments. J Biol Chem 276:2088±2097, 2001 Rothnagel JA, Dominey AM, Dempsey LD, et al: Mutations in the rod domain of keratin 1 and keratin 10 in epidermolytic hyperkeratosis. Science 257:1128± 1130, 1992 Strelkov SV, Herrmann H, Geisler N, Wedig T, Zimbelmann R, Aebi U, Burkhard P: Conserved segments 1A and 2B of the intermediate ®lament dimer: their atomic structure and role in ®lament assembly. EMBO J 21:1255±1266, 2002 Whittock NV, Ashton GHS, Grif®ths WAD, Eady RAJ, McGrath JA: New mutations in keratin 1 that cause bullous congenital ichthyosiform erythroderma and keratin 2e that cause ichthyosis bullosa of Siemens. Br J Dermatol 145:330±335, 2001 Yang J-M, Chipev CC, DiGiovanna JJ, Bale SJ, Marekov LN, Steinert PM, Compton JG: Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis. J Invest Dermatol 102:17±23, 1994 Yang J-M, Nam K, Kim H-C, Lee J-H, Park J-K, Wu K, Lee E-S, Steinert PM: A novel glutamic acid to aspartic acid mutation near the end of the 2B rod domain in the keratin 1 chain in epidermolytic hyperkeratosis. J Invest Dermatol 112:376±379, 1999
Two Novel Mutations in the Keratin 1 Gene in Epidermolytic Hyperkeratosis the KRT2e gene was reported in ichthyosis bullosa of Siemens (Whittock et al, 2001). In patient EHK-AS, we found a single nucleotide substitution on one allele of codon 475 in exon 7 of the KRT1 gene (CTG to CCG) so that the sequence gel of Fig 1(b) shows both a wild-type T and a mutant C at nucleotide position 1457 (GenBank NM-006121; relative to the translation start point) in the proband. The mutant allele encodes a proline residue instead of a wild-type leucine residue in position 113 of 2B rod domain segment. This nucleotide substitution destroys an MspI restriction enzyme site (C/CGG), which was used to con®rm that the unaffected parents and 50 other unrelated unaffected persons do not posses this new mutation.
To the Editor: Epidermolytic hyperkeratosis (EHK, MIM113800) is a rare autosomal dominant genodermatosis, although many cases occur sporadically as new mutations. It is characterized by blistering and erythema at birth, and development of hyperkeratosis with increasing age. Ultrastructural analyses reveal clumping of the keratin intermediate ®laments (KIF) within suprabasal keratinocytes of the epidermis. Previous studies have demonstrated that EHK is caused by mutations in the genes encoding the keratin 1 (K1) or the K10 proteins (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992; McLean and Lane, 1995; Korge and Krieg, 1996). Clinically, EHK has been divided into the palmoplantar type (palm sole, PS) and the nonpalmoplantar type (nonpalm sole), and each type has been subdivided into three subtypes according to the nature and severity of scaling (DiGiovanna and Bale, 1994; DiGiovanna et al, 1998). The mutation/clinical phenotype correlations available suggest that the PS and nonpalm sole types are due primarily to mutations in the KRT1 and KRT10 genes, respectively. The more severe PS-1 and PS-2 cases are caused by mutations that change sequences encoding the beginning of the 1A rod domain segment, or towards the end of the 2B rod domain segment (Yang et al, 1994, 1999; Arin et al, 1999, 2000; Cserhalmi-Friedman et al, 2000). In this study, we describe two separate cases of EHK, classi®ed as PS-2, caused by two different novel mutations: Asn187Lys located in exon 1, which is the 8th residue of the 1A rod domain segment; and Leu475Pro located in exon 7, which is the 10th residue from the end of 2B rod domain segment of K1. Two unrelated children, EHK-SJ and EHK-AS, have diffuse thick keratoderma on both palms and soles as well as moderate scaly patches on their entire bodies, but the morphology of their hair, nails, and teeth are normal. Light microscopic examination revealed vacuolar degeneration in the spinous and granular layers. As both sets of parents were normal, these represent sporadic cases. Both were classi®ed as having the PS-2 form of EHK. DNA was extracted (Chipev et al, 1994; Yang et al, 1994) from both the affected and unaffected family members, and from 50 unrelated normal individuals used as controls. Following sequencing of all exons of the KRT1 and KRT10 genes, patient EHK-SJ had a single nucleotide substitution on one allele of codon 187 in exon 1 of the KRT1 gene (AAC to AAA) so that the sequence gel of Fig 1(a) shows both the wild-type C and a mutant A at nucleotide position 564 (GenBank NM-006121; relative to the translation start point) in the proband. The mutant allele encodes a lysine residue instead of a wild-type asparagine residue in position 8 of the 1A rod domain segment. As this nucleotide substitution does not create or destroy a restriction enzyme site, we performed a polymerase chain reaction ampli®cation of a speci®c allele assay (Yang et al, 1994, 1999). As the parents and 50 other unrelated normal controls do not have this substitution, it represents a new mutation. The same mutation in Manuscript received June 7, 2002; accepted for publication June 10, 2002 Reprint requests to: June-Mo Yang, MD, PhD, Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, 50 Il-won Dong, Kangnam Ku, Seoul, 135-710, Republic of Korea. Email: [email protected] 0022-202X/02/$15.00
Figure 1. (a) EHK-SJ. DNA sequences showing C to A change (arrow), which introduces an Asn187Lys amino acid substitution in the 1A rod domain segment of K1. (b)EHK-AS. DNA sequences showing T to C change (arrow), which introduces a Leu475Pro amino acid substitution in the 2B rod domain segment of K1.
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 976
VOL. 119, NO. 4 OCTOBER 2002
LETTERS TO THE EDITOR
977
Next, we synthesized four 18-residue peptides corresponding to the beginning of the 1A and the end of 2B region of wild-type K1, or sequences with the described amino acid substitutions, respectively. These were used for an assay that uses synthetic peptides to interfere with KIF assembly (Chipev et al, 1992): wild-type peptides disassemble KIF, whereas peptides bearing identi®ed substitutions caused by novel mutations often do not. In both cases, we found that the wild-type 1A or 2B peptides interfered with the formation of KIF in vitro; however, neither mutant peptide interfered with KIF assembly (Fig 2). From this we can conclude that the substitutions resulting from the observed mutations affect normal KIF structure. The three-dimensional structures of the 1A (Strelkov et al, 2002) and 2B (Herrmann et al, 2000) domains of the related IF protein vimentin have been solved. As the amino acid sequences of vimentin and K1 are conserved in these regions, their structures are likely to be similar. An asparagine residue in position 8 of the 1A domain has been conserved in all IF proteins. Its side chain projects laterally outward from the axis of the coiled-coil, where it presumably interacts with residues on an adjacent molecule. Indeed, we have predicted that this residue forms H-bonds with residues in the L2 linker region in the A11 alignment mode of two adjacent molecules in the tetramer (Mehrani et al, 2001). By model building analyses, the substitution of a larger positively charged lysine residue will interfere with such interactions. Thus this Asn187Lys substitution in patient EHK-SJ will likely severely interfere with KIF structure at the twomolecule hierarchical stage of KIF assembly. Likewise, a leucine residue in position 113 of the 2B rod domain segment has been conserved in all IF chains. Based on the extant model of this region in vimentin, this leucine residue marks the point at which the coiledcoil 2B sequences unravel and fold back along the axis of the coiledcoil. Insertion of an aberrant cis peptide bond of a proline residue at this point will prevent this refold. Accordingly, the Leu475Pro substitution will cause failed KIF assembly at the two-molecule level of assembly. We thank the families for their participation and cooperation, and Dr U. Aebi for sharing the structure of vimentin before publication. This work was supported by a grant (09-PJ3-PG6-01GN12-0001) of the 2001 Good Health R & D Project, Ministry of Health and Welfare, Republic of Korea (Yang).
Dong-Youn Lee,*² Kwang-Sung Ahn,² Cha-Hui Lee,² NarkKyoung Rho,* Joo-Heung Lee,* Eil-Soo Lee,* Peter M. Steinert,³ and Jun-Mo Yang*² *Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, ²Clinical Research Center, Samsung Biomedical Research Institute, Seoul, Republic of Korea, ³Laboratory of Skin Biology, National Institutes of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, U.S.A. REFERENCES Arin MJ, Longley MA, Kuster W, Huber M, Hohl D, Rothnagel JA, Roop DR: An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis. Exp Dermatol 8:124±127, 1999 Arin MJ, Longley MA, Epstein EH, Rothnagel JA, Roop DR: Identi®cation of a novel mutation in keratin 1 in a family with epidermolytic hyperkeratosis. Exp Dermatol 9:16±19, 2000 Cheng J, Snyder AJ, Yu Q-C, Paller AS, Fuchs E: The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-speci®c genes. Cell 70:811±819, 1992 Chipev CC, Korge BP, Markova N, Bale SJ, DiGiovanna JJ, Compton JG, Steinert PM: A leucine-proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 70:821±828, 1992 Chipev CC, Yang J-M, DiGiovanna JJ, Steinert PM, Marekov LN, Compton JG, Bale SJ: Preferential sites in keratin 10 that are mutated in epidermolytic hyperkeratosis. Am J Hum Genet 54:179±190, 1994 Cserhalmi-Friedman PB, Squeo R, Gordon D, Garzon M, Schneiderman P, Grossman ME, Christiano AM: Epidermolyic hyperkeratosis in a Hispanic family resulting from a mutation in the keratin 1 gene. Clin Exp Dermatol 25:241±243, 2000
Figure 2. KIF disassembly assay using 18-residue wild-type 1A (b) or 2B (d) peptides. In both cases, normal KIF (a) are disassembled into small particles within 30 min at 23°; however, the Asn187Lys mutant 1A peptide (c) or Leu475Pro mutant 2B peptide (e) had no discernible effect on preformed KIF, or KIF assembly in vitro (not shown). The assay suggests that the structure of the mutant portions of the proteins will interfere with KIF assembly. Scale bar = 200 nm. DiGiovanna JJ, Bale SJ: Clinical heterogeneity in epidermoltyic hyperkeratosis. Arch Dermatol 130:1026±1035, 1994 DiGiovanna JJ, Bale SJ, Steinert PM: Epidermolytic hyperkeratosis. In: Kupper T (ed.). Textbook of Molecular Medicine, Part 8, Molecular Mechanisms in Skin and Connective Tissues. Totowa: Human Press, 1998, 707±712 Herrmann H, Strelkov S, Feja B, et al: The intermediate ®lament protein consensus motif of helix 2B: atomic structure and contribution to assembly. J Mol Biol 298:817±832, 2000 Korge BP, Krieg T: The molecular basis for inherited bullous diseases. J Mol Med 74:59±70, 1996 McLean WHI, Lane EB: Intermediate ®laments in disease. Curr Opin Cell Biol 7:118± 125, 1995 Mehrani T, Morasso MI, Marekov LN, Parry DAD, Steinert PM: Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate ®laments. J Biol Chem 276:2088±2097, 2001 Rothnagel JA, Dominey AM, Dempsey LD, et al: Mutations in the rod domain of keratin 1 and keratin 10 in epidermolytic hyperkeratosis. Science 257:1128± 1130, 1992 Strelkov SV, Herrmann H, Geisler N, Wedig T, Zimbelmann R, Aebi U, Burkhard P: Conserved segments 1A and 2B of the intermediate ®lament dimer: their atomic structure and role in ®lament assembly. EMBO J 21:1255±1266, 2002 Whittock NV, Ashton GHS, Grif®ths WAD, Eady RAJ, McGrath JA: New mutations in keratin 1 that cause bullous congenital ichthyosiform erythroderma and keratin 2e that cause ichthyosis bullosa of Siemens. Br J Dermatol 145:330±335, 2001 Yang J-M, Chipev CC, DiGiovanna JJ, Bale SJ, Marekov LN, Steinert PM, Compton JG: Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis. J Invest Dermatol 102:17±23, 1994 Yang J-M, Nam K, Kim H-C, Lee J-H, Park J-K, Wu K, Lee E-S, Steinert PM: A novel glutamic acid to aspartic acid mutation near the end of the 2B rod domain in the keratin 1 chain in epidermolytic hyperkeratosis. J Invest Dermatol 112:376±379, 1999