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Novel keratin 5 mutations in epidermolysis bullosa simplex: Cases with unusual genotype–phenotype correlation
Journal of Dermatological Science (2007) 48, 229—232
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Novel keratin 5 mutations in epidermolysis bullosa simplex: Cases with unusual genotype—phenotype correlation Epidermolysis bullosa simplex (EBS) is a group of hereditary skin diseases characterized by blistering from mild trauma as a result of basal epidermal cells cytolysis. EBS is commonly inherited in an autosomal dominant fashion due to mutations in cytokeratin genes 5 (K5) and 14 (K14). It is subdivided into three major subtypes according to symptom severity. EBS Dowling-Meara (EBS-DM) is the most severe phenotype, characterized by herpetiform blisters on the whole body and hyperkeratosis of the palms and soles. Electron microscopically, keratin filament clumping is a pathognomonic sign of EBS-DM. EBS Koebner (EBS-K) has milder generalized blistering, whereas EBS Weber-Cockayne (EBS-WC) is the mildest, with blistering confined to the hands and feet [1]. Keratins are diverse structural proteins that polymerize to intermediate filaments. A characteristic feature of filament proteins is division of the central a-helical rod domain into four segments (1A, 1B, 2A, and 2B) by three short non-helical linker domains (L1, L12, and L2) [2]. Mutations responsible for EBSDM lie within the highly conserved ends of the rod domain (1A, 2B), critical for K5 and K14 assembly. On the other hand, EBS-K mutations are diffusely scattered within rod domains and in the L12 linker domains of both keratin genes, whereas EBS-WC mutations are mostly within non-helical regions [1]. We performed gene analysis in patients with EBS. After informed consent, genomic DNA was extracted from peripheral blood lymphocytes using a DNA extraction kit (Qiagen). All exons of K5 and K14 genes were amplified by polymerase chain reaction (PCR) according to a previous report [1]. Sequence analyses were performed using Big Dye terminator technology (ABI 3100 Perkin-Elmer). Genomic DNA from 50 normal, healthy, Koreans were used as controls. To assess potential pathogenicity of the mutations found in this study, information theory
splice site analysis was carried out via the Internet interface at http://splice.cmh.edu/ [3]. A 27-year-old Korean female presented with history of blister formation since birth, mainly on the soles and palms, and occasionally the trunk (Fig. 1a— c). The soles were hyperkeratotic, but without mucous membrane involvement. Her parents were clinically normal. Histological examination and immunofluorescence mapping revealed primary separation within the basal cell layer. No keratin clumping was detected in the electron microscopy. We diagnosed her as EBS-K, and performed mutation analysis of the K5 and K14 genes. Direct sequencing of patient 1 revealed G-to-C transition at nucleotide 1398 in exon 7 (Fig. 1d) of the K5 gene. This transition results in replacement of glutamic acid with aspartic acid in codon 466 (p.E466D). No such mutation was found in the 50 unrelated controls. A 2-month-old Korean male presented with generalized blistering (Fig. 1e and f). Blisters healed without scarring and eyes, nails, and mucous membranes were not involved. His parents were clinically normal. Histological examination and immunofluorescence mapping of a trunk skin specimen revealed primary separation within the basal cell layer. Under electron microscopy, no definite keratin clumping was seen, but some aggregation was noted (Fig. 1g). Based on clinical and microscopic examinations, he was diagnosed EBS-K. On the mutational analysis, T-to-C transition at nucleotide 449 in exon 1 (Fig. 1h) of the K5 gene was detected. This transition results in replacement leucine with proline in codon 150 (p.L150P). No such mutation was found in the 50 unrelated controls. Computer analysis was done to assess potential for each mutation to affect mRNA splicing. No information contents score (Ri) change was predicted for the mutation found in this study. Each keratin protein has a similar structure, possessing a 310-amino acid residue central a-helical rod domain. Sequences at either end of the rod domain, termed helix initiation peptides (HIP) or helix termination peptides (HTP), respectively, are highly conserved and critical for intermediate filaments assembly. In addition, the region between
0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.07.014
230
Letter to the Editor
Fig. 1 Clinical features, electron microscopic findings, and direct sequencing data of patients 1 (a—d) and 2 (e—h). (a— c) In patient 1, multiple blisters and erosion were found on the palms, soles, and occasionally the trunk. (d) Direct PCR product sequencing revealed G-to-C transition at nucleotide 1398 (c.1398G > C, p.E466D) in exon 5 of the K5 gene. (e and f) In patient 2, blisters and erosions were present on the whole body. Herpetiform blisters were noted on the trunk. (g) Electron microscopic examination of patient 2 revealed cytolysis in the basal cell (asterisk). There was no definite keratin filament clumping, but some aggregation was noted (white arrow). (h) Direct PCR product sequencing revealed T-to-C transition at nucleotide 449 (c.449G > C, p.L150P) in exon 1 of the K5 gene.
residues 100 and 113 of the 2B rod domain segment is known as a coiled-coil trigger motif, and is absolutely essential for molecular stability and intermediate filament assembly [2]. A mutation within this motif, such as p.I467T [4], is known to induce a severe phenotype, such as EBS-DM. However, in this study, although mutation p.E466D is located in this motif, it only caused a mild phenotype. It is quite unusual that a mutation in HTP induces a mild phenotype. There is only one other mutation in HTP with non-EBS-DM phenotype (p.L463P) [5,6]. Most mutations causing EBS-DM are located in amino acid sequences from 467 to 477 (Table 1). Moreover, most mutations causing EBS-DM change their side chain polarity or acidity, while there is no change in the mutations, p.L463P and p.E466D (Table 1) Therefore, we can speculate that amino acid sequences from 467 to 477 are more important than those of proximal positions, or that the side chain polarity and acidity are more critical to the struc-
ture formation of the keratin 5. However, these must be more fully elucidated. The a-helical rod domain possesses a characteristic heptad structure (abcdefg)n, which has apolar interactions between positions e and g, and salt bridges between positions a and d [7]. It has been suggested that mutations affecting heptad positions a, d, e, and g are associated with a severe phenotype [8]. In patient 1, the amino acid p.E466D corresponds to heptad position g (Table 1). However, she did not show a severe phenotype. Actually, when we reviewed reports of mutations in HTP of K5 gene, we did not find any significant correlation between mutation heptad position and phenotype (Table 1). Moreover, Smith et al. [7] described that mutations in positions a and d, were not always of structural significance. Therefore, it seems that another factor influences genotype—phenotype correlation, other than heptad position.
Letter to the Editor
231
Table 1 Relative position of the mutation in 2B domain of K5 gene and their heptad position Amino
Heptad
Amino
Mutation
The changes in side chain polarity or acidity
acid
position
acid
461
b
L
462
c
A
463
d
L
p.L463P
Non-polar neutral → nonpolar neutral (no change)
464
e
D
465
f
V
466
g
E
p.E466D
Polar acidic → polar acidic (no change)
467
a
I
p.I467T
Non-polar neutral → polar neutral
468
b
A
469
c
T
p.T469P
Polar neutral → nonpolar neutral
470
d
Y
471
e
R
472
f
K
p.K472X
Polar basic → premature termination codon
473
g
L
474
a
L
475
b
E
p.E475K
Polar acidic → polar basic
476
c
G
477
d
E
p.E477K
Polar acidic → polar basic
p.E477X
Polar acidic → premature termination codon
position
478
e
E
479
f
C
480
g
R
481
a
L
Blue letter: the mutation in the patient with EBS-K; red letter: the mutation in the patient with EBS-DM.
The mutation, p.L150P, also shows unusual genotype—phenotype correlation. The proband shows generalized blisters despite the mutation being in the non-helical head region, which is usually associated with EBS-WC. There are two missense mutations in patients with EBS-K and one splicing site mutation in EBS-DM which is located in the H1 region [1,9,10]. We ran a computer analysis to assess potential for the mutation to affect splicing, but no possible alternative splicing was detected. It is unusual for an EBS-K mutation to be located in the
H1 region. Moreover, the proband shows a more severe phenotype than patients with common EBS-K, in spite of the lack of keratin clumping in our patient. This is the first missense mutation report of a severe EBS phenotype in the H1 domain of the K5 gene. In conclusion, we report two novel mutations, p.L150P and p.E466D, in the K5 gene, which show unusual genotype—phenotype correlation. The exact effect of the mutations needs to be more fully elucidated.
232
Conflict of interest The authors state no conflict of interest.
References [1] Yasukawa K, Sawamura D, Goto M, Nakamura H, Jung SY, Kim SC, et al. Epidermolysis bullosa simplex in Japanese and Korean patients: genetic studies in 19 cases. Br J Dermatol 2006;155:313—7. [2] Wu KC, Bryan JT, Morasso MI, Jang SI, Lee JH, Yang JM, et al. Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments. Mol Biol Cell 2000;11:3539—58. [3] Nalla VK, Rogan PK. Automated splicing mutation analysis by information theory. Hum Mutat 2005;25:334—42. [4] Irvine AD, McKenna KE, Bingham A, Nevin NC, Hughes AE. A novel mutation in the helix termination peptide of keratin 5 causing epidermolysis bullosa simplex Dowling-Meara. J Invest Dermatol 1997;109:815—6. [5] Nomura K, Umeki K, Meng X, Tamai K, Sawamura D, Hosokawa M, et al. A keratin K5 mutation (Leu 463!Pro) in a family with the Weber-Cockayne type of epidermolysis bullosa simplex. Arch Dermatol Res 1997;289:493—5. [6] Dong W, Ryynanen M, Uitto J. Identification of a leucine-toproline mutation in the keratin 5 gene in a family with the generalized Koebner type of epidermolysis bullosa simplex. Hum Mutat 1993;2:94—102. [7] Smith TA, Steinert PM, Parry DA. Modeling effects of mutations in coiled-coil structures: case study using epidermolysis bullosa simplex mutations in segment 1a of K5/K14 intermediate filaments. Proteins 2004;55:1043—52. [8] Muller FB, Kuster W, Wodecki K, Almeida HJ, BrucknerTuderman L, Krieg T, et al. Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa
Letter to the Editor simplex: implications for disease phenotype and keratin filament assembly. Hum Mutat 2006;27:719—20. [9] Saeki H, Nakamura K, Tsunemi Y, Komine M, Tamaki K. Novel mutation (Asp158Val) in H1 domain of keratin 5 gene in a Japanese patient with Koebner-type epidermolysis bullosa simplex. J Dermatol 2006;33:692—5. [10] Rugg EL, Rachet-Prehu MO, Rochat A, Barrandon Y, Goossen M, Lane EB, et al. Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex. Eur J Hum Genet 1999;7:293—300.
Se-Woong Oh Jeong Sun Lee Moon Young Kim Soo-Chan Kim* Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea *Corresponding author at: Department of Dermatology, Yonsei University College of Medicine, Yongdong Severance Hospital, 146-92, Dogok-dong, Kangnam-gu, Seoul 135-720, Republic of Korea. Tel.: +82 2 2019 3360; fax: +82 2 3463 6136 E-mail address: [email protected] (S.-C. Kim) 22 June 2007
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Novel keratin 5 mutations in epidermolysis bullosa simplex: Cases with unusual genotype—phenotype correlation Epidermolysis bullosa simplex (EBS) is a group of hereditary skin diseases characterized by blistering from mild trauma as a result of basal epidermal cells cytolysis. EBS is commonly inherited in an autosomal dominant fashion due to mutations in cytokeratin genes 5 (K5) and 14 (K14). It is subdivided into three major subtypes according to symptom severity. EBS Dowling-Meara (EBS-DM) is the most severe phenotype, characterized by herpetiform blisters on the whole body and hyperkeratosis of the palms and soles. Electron microscopically, keratin filament clumping is a pathognomonic sign of EBS-DM. EBS Koebner (EBS-K) has milder generalized blistering, whereas EBS Weber-Cockayne (EBS-WC) is the mildest, with blistering confined to the hands and feet [1]. Keratins are diverse structural proteins that polymerize to intermediate filaments. A characteristic feature of filament proteins is division of the central a-helical rod domain into four segments (1A, 1B, 2A, and 2B) by three short non-helical linker domains (L1, L12, and L2) [2]. Mutations responsible for EBSDM lie within the highly conserved ends of the rod domain (1A, 2B), critical for K5 and K14 assembly. On the other hand, EBS-K mutations are diffusely scattered within rod domains and in the L12 linker domains of both keratin genes, whereas EBS-WC mutations are mostly within non-helical regions [1]. We performed gene analysis in patients with EBS. After informed consent, genomic DNA was extracted from peripheral blood lymphocytes using a DNA extraction kit (Qiagen). All exons of K5 and K14 genes were amplified by polymerase chain reaction (PCR) according to a previous report [1]. Sequence analyses were performed using Big Dye terminator technology (ABI 3100 Perkin-Elmer). Genomic DNA from 50 normal, healthy, Koreans were used as controls. To assess potential pathogenicity of the mutations found in this study, information theory
splice site analysis was carried out via the Internet interface at http://splice.cmh.edu/ [3]. A 27-year-old Korean female presented with history of blister formation since birth, mainly on the soles and palms, and occasionally the trunk (Fig. 1a— c). The soles were hyperkeratotic, but without mucous membrane involvement. Her parents were clinically normal. Histological examination and immunofluorescence mapping revealed primary separation within the basal cell layer. No keratin clumping was detected in the electron microscopy. We diagnosed her as EBS-K, and performed mutation analysis of the K5 and K14 genes. Direct sequencing of patient 1 revealed G-to-C transition at nucleotide 1398 in exon 7 (Fig. 1d) of the K5 gene. This transition results in replacement of glutamic acid with aspartic acid in codon 466 (p.E466D). No such mutation was found in the 50 unrelated controls. A 2-month-old Korean male presented with generalized blistering (Fig. 1e and f). Blisters healed without scarring and eyes, nails, and mucous membranes were not involved. His parents were clinically normal. Histological examination and immunofluorescence mapping of a trunk skin specimen revealed primary separation within the basal cell layer. Under electron microscopy, no definite keratin clumping was seen, but some aggregation was noted (Fig. 1g). Based on clinical and microscopic examinations, he was diagnosed EBS-K. On the mutational analysis, T-to-C transition at nucleotide 449 in exon 1 (Fig. 1h) of the K5 gene was detected. This transition results in replacement leucine with proline in codon 150 (p.L150P). No such mutation was found in the 50 unrelated controls. Computer analysis was done to assess potential for each mutation to affect mRNA splicing. No information contents score (Ri) change was predicted for the mutation found in this study. Each keratin protein has a similar structure, possessing a 310-amino acid residue central a-helical rod domain. Sequences at either end of the rod domain, termed helix initiation peptides (HIP) or helix termination peptides (HTP), respectively, are highly conserved and critical for intermediate filaments assembly. In addition, the region between
0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.07.014
230
Letter to the Editor
Fig. 1 Clinical features, electron microscopic findings, and direct sequencing data of patients 1 (a—d) and 2 (e—h). (a— c) In patient 1, multiple blisters and erosion were found on the palms, soles, and occasionally the trunk. (d) Direct PCR product sequencing revealed G-to-C transition at nucleotide 1398 (c.1398G > C, p.E466D) in exon 5 of the K5 gene. (e and f) In patient 2, blisters and erosions were present on the whole body. Herpetiform blisters were noted on the trunk. (g) Electron microscopic examination of patient 2 revealed cytolysis in the basal cell (asterisk). There was no definite keratin filament clumping, but some aggregation was noted (white arrow). (h) Direct PCR product sequencing revealed T-to-C transition at nucleotide 449 (c.449G > C, p.L150P) in exon 1 of the K5 gene.
residues 100 and 113 of the 2B rod domain segment is known as a coiled-coil trigger motif, and is absolutely essential for molecular stability and intermediate filament assembly [2]. A mutation within this motif, such as p.I467T [4], is known to induce a severe phenotype, such as EBS-DM. However, in this study, although mutation p.E466D is located in this motif, it only caused a mild phenotype. It is quite unusual that a mutation in HTP induces a mild phenotype. There is only one other mutation in HTP with non-EBS-DM phenotype (p.L463P) [5,6]. Most mutations causing EBS-DM are located in amino acid sequences from 467 to 477 (Table 1). Moreover, most mutations causing EBS-DM change their side chain polarity or acidity, while there is no change in the mutations, p.L463P and p.E466D (Table 1) Therefore, we can speculate that amino acid sequences from 467 to 477 are more important than those of proximal positions, or that the side chain polarity and acidity are more critical to the struc-
ture formation of the keratin 5. However, these must be more fully elucidated. The a-helical rod domain possesses a characteristic heptad structure (abcdefg)n, which has apolar interactions between positions e and g, and salt bridges between positions a and d [7]. It has been suggested that mutations affecting heptad positions a, d, e, and g are associated with a severe phenotype [8]. In patient 1, the amino acid p.E466D corresponds to heptad position g (Table 1). However, she did not show a severe phenotype. Actually, when we reviewed reports of mutations in HTP of K5 gene, we did not find any significant correlation between mutation heptad position and phenotype (Table 1). Moreover, Smith et al. [7] described that mutations in positions a and d, were not always of structural significance. Therefore, it seems that another factor influences genotype—phenotype correlation, other than heptad position.
Letter to the Editor
231
Table 1 Relative position of the mutation in 2B domain of K5 gene and their heptad position Amino
Heptad
Amino
Mutation
The changes in side chain polarity or acidity
acid
position
acid
461
b
L
462
c
A
463
d
L
p.L463P
Non-polar neutral → nonpolar neutral (no change)
464
e
D
465
f
V
466
g
E
p.E466D
Polar acidic → polar acidic (no change)
467
a
I
p.I467T
Non-polar neutral → polar neutral
468
b
A
469
c
T
p.T469P
Polar neutral → nonpolar neutral
470
d
Y
471
e
R
472
f
K
p.K472X
Polar basic → premature termination codon
473
g
L
474
a
L
475
b
E
p.E475K
Polar acidic → polar basic
476
c
G
477
d
E
p.E477K
Polar acidic → polar basic
p.E477X
Polar acidic → premature termination codon
position
478
e
E
479
f
C
480
g
R
481
a
L
Blue letter: the mutation in the patient with EBS-K; red letter: the mutation in the patient with EBS-DM.
The mutation, p.L150P, also shows unusual genotype—phenotype correlation. The proband shows generalized blisters despite the mutation being in the non-helical head region, which is usually associated with EBS-WC. There are two missense mutations in patients with EBS-K and one splicing site mutation in EBS-DM which is located in the H1 region [1,9,10]. We ran a computer analysis to assess potential for the mutation to affect splicing, but no possible alternative splicing was detected. It is unusual for an EBS-K mutation to be located in the
H1 region. Moreover, the proband shows a more severe phenotype than patients with common EBS-K, in spite of the lack of keratin clumping in our patient. This is the first missense mutation report of a severe EBS phenotype in the H1 domain of the K5 gene. In conclusion, we report two novel mutations, p.L150P and p.E466D, in the K5 gene, which show unusual genotype—phenotype correlation. The exact effect of the mutations needs to be more fully elucidated.
232
Conflict of interest The authors state no conflict of interest.
References [1] Yasukawa K, Sawamura D, Goto M, Nakamura H, Jung SY, Kim SC, et al. Epidermolysis bullosa simplex in Japanese and Korean patients: genetic studies in 19 cases. Br J Dermatol 2006;155:313—7. [2] Wu KC, Bryan JT, Morasso MI, Jang SI, Lee JH, Yang JM, et al. Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments. Mol Biol Cell 2000;11:3539—58. [3] Nalla VK, Rogan PK. Automated splicing mutation analysis by information theory. Hum Mutat 2005;25:334—42. [4] Irvine AD, McKenna KE, Bingham A, Nevin NC, Hughes AE. A novel mutation in the helix termination peptide of keratin 5 causing epidermolysis bullosa simplex Dowling-Meara. J Invest Dermatol 1997;109:815—6. [5] Nomura K, Umeki K, Meng X, Tamai K, Sawamura D, Hosokawa M, et al. A keratin K5 mutation (Leu 463!Pro) in a family with the Weber-Cockayne type of epidermolysis bullosa simplex. Arch Dermatol Res 1997;289:493—5. [6] Dong W, Ryynanen M, Uitto J. Identification of a leucine-toproline mutation in the keratin 5 gene in a family with the generalized Koebner type of epidermolysis bullosa simplex. Hum Mutat 1993;2:94—102. [7] Smith TA, Steinert PM, Parry DA. Modeling effects of mutations in coiled-coil structures: case study using epidermolysis bullosa simplex mutations in segment 1a of K5/K14 intermediate filaments. Proteins 2004;55:1043—52. [8] Muller FB, Kuster W, Wodecki K, Almeida HJ, BrucknerTuderman L, Krieg T, et al. Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa
Letter to the Editor simplex: implications for disease phenotype and keratin filament assembly. Hum Mutat 2006;27:719—20. [9] Saeki H, Nakamura K, Tsunemi Y, Komine M, Tamaki K. Novel mutation (Asp158Val) in H1 domain of keratin 5 gene in a Japanese patient with Koebner-type epidermolysis bullosa simplex. J Dermatol 2006;33:692—5. [10] Rugg EL, Rachet-Prehu MO, Rochat A, Barrandon Y, Goossen M, Lane EB, et al. Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex. Eur J Hum Genet 1999;7:293—300.
Se-Woong Oh Jeong Sun Lee Moon Young Kim Soo-Chan Kim* Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea *Corresponding author at: Department of Dermatology, Yonsei University College of Medicine, Yongdong Severance Hospital, 146-92, Dogok-dong, Kangnam-gu, Seoul 135-720, Republic of Korea. Tel.: +82 2 2019 3360; fax: +82 2 3463 6136 E-mail address: [email protected] (S.-C. Kim) 22 June 2007