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Pachyonychia congenita type 2: Keratin 17 mutation in a Japanese case
Journal of the American Academy of Dermatology Volume 38, Number 6, Part 1
Brief communications 1007
Pachyonychia congenita type 2: Keratin 17 mutation in a Japanese case Wataru Fujimoto, MD,a Gen Nakanishi, MD,a Satoshi Hirakawa, MD,a Tohru Nakanishi, PhD,b Tsuyoshi Shimo, DDS,b Masaharu Takigawa, DDS, PhD,b and Jirô Arata, MDa Okayama, Japan Pachyonychia congenita (PC) is an autosomal dominant genodermatosis first described by Muller.1 Two major subtypes of PC have been described.2 Jadassohn-Lewandowsky type PC (MIM #167200; PC1) is characterized by onychogryphosis; hyperkeratosis of the palms, soles, knees, and elbows; follicular hyperkeratosis; and leukoplakia of the oral mucous membranes.3 Jackson-Lawler type PC (MIM #167210; PC2) shows no oral leukoplakia but is characterized by natal teeth and multiple cutaneous cysts and pachyonychia.4 Histopathologic condition of hyperkeratotic skin shows moderate to marked hyperkeratosis and acanthosis. Ultrastructural analysis of keratinocytes in the stratum spinosum of affected areas shows large tonofibril aggregates without signs of cellular disruption or cell death.5,6 The ultrastructural finding of aggregated keratin filament bundles in keratinocytes is a common feature of keratin diseases such as epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic palmoplantar keratoderma, and ichthyosis bullosa of Siemens. In these genetic skin disorders, mutations of keratin genes cause dysfunction of keratin intermediate filaments, leading to blister formation or hyperkeratotic skin lesions.7-10 Heterozygous missense mutations of the keratin 16 gene and keratin 17 (K17) gene have been identified in PC1 and PC2, respectively.6,11 A heterozygous keratin 6a deletion has also been identified in a PC1 family.12 In the present study we report a missense mutation within the helix ini-
From the Department of Dermatology, Okayama University Medical School,a and the Department of Biochemistry and Molecular Dentistry, Okayama University Dental School.b Reprint requests: Wataru Fujimoto, MD, Department of Dermatology, Okayama University Medical School, 2-5-1 Shikata, Okayama 700, Japan. J Am Acad Dermatol 1998;38:1007-9. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/54/89794
tiation motif of the K17 gene in a sporadic Japanese case of Jackson-Lawler type PC. PATIENT AND METHODS
Patient The pedigree examined in this study is shown in Fig. 1, A. The family history of the patient showed no consanguinity and no relatives with similar conditions. The patient is a 24-year-old Japanese man who at birth was covered with white, milia-like cysts (Fig. 1, B) and had two natal teeth. All of his nails were unusually thick from an early age. At 5 or 6 years of age, cysts developed on his face and extremities; the number of cysts gradually increased on his buttocks, groin, and axillae. At 12 or 13 years of age, these cysts became painful, enlarged, and infected. He underwent numerous surgical procedures for infected cysts in the axillae and was diagnosed with hidradenitis suppurativa. Physical examination at the time of the present study revealed numerous cysts and nodules of various sizes all over the body (Fig. 1, C), pachyonychia on all fingers and toes (Fig. 1, D), pili torti (Fig. 1, E), and callus-like focal plantar keratoderma on the left sole. The mouth, tongue, and teeth were normal. Biopsy specimens of several cysts suggested a diagnosis of steatocystoma multiplex. Immunohistochemistry showed positive staining for K17 in the suprabasal lining cells of the cyst wall (Fig. 1, F).
Reverse transcription–polymerase chain reaction and DNA sequencing Because K17 has pseudogenes,13 we performed reverse transcription–polymerase chain reaction to avoid amplifying these genes from genomic DNA. Total RNA was extracted from the patient’s steatocystomas because K17 is abundantly expressed in epithelial cells of the cysts (Fig. 1, F).14 As a control, total RNA was extracted from involved lesions of several patients with psoriasis, because K17 is expressed in abundance in psoriatic lesions.15 The total RNA was reverse transcribed, and the cDNA encoding the entire rod domain of K17 was amplified by PCR with primers k17p3 and k17p6, as described previously,6 and sequenced.
Journal of the American Academy of Dermatology June 1998
1008 Brief communications
A
D
B
C
E
F
Fig. 1. Pedigree of the PC-2 family, clinical photographs of proband and expression of K17 in steatocystoma. A, Family PC-2, sporadic case of JacksonLawler PC. DNA was obtained from four family members (*) including proband (arrow). B, Numerous white, milia-like cysts were seen when the patient was 1 month of age. C, Fresh-colored papules and nodules on anterior chest. D, Thickened fingernails. E, Helical body hair on lower leg. F, Expression of K17 in the lining cells of steatocystoma. Immunohistochemistry was performed on paraffin sections boiled in 10 mmol/L citrate buffer reacted with monoclonal anti-cytokeratin 17, E3 (M7046; Dako, A/S, Denmark).
Allele-specific PCR analysis Allele-specific PCR (AS-PCR) was performed to screen the mutation of subclones containing the 267 base pairs (bp) fragment. The normal allele-specific primer, K17pN (5´-GCCACCATGCAGAACCTCAA3´), and a mutant allele-specific primer, K17pM (5´GCCACCATGCAGAACCTCAG-3´), were used as forward primers, and K17p12 (5´-GCCGGATGTTGGCATTGTC-3´) was used as a common reverse primer. AS-PCR was also performed to screen the mutations in genomic DNAs. K17pN and K17pM were used as the forward primers; K17p10 was used as the reverse primer.6 RESULTS
Sequence analysis of K17 Direct sequencing of amplified 1032 fragments from the patient’s cDNA disclosed an unreadable base in the helix initiation motif of the K17 gene. By subcloning the 267 bp fragment in the helix initiation motif and sequencing some of the positive clones, we identified A to G transition at position 423 in some clones thought to be derived from mutant allele of the patient (Fig. 2, A). This transition results in an asparagine (N) to serine (S) substitution. Other clones showed a normal sequence
A
B Fig. 2. Identification of missense mutation N92S in the K17 gene. A, Nucleotide sequencing of subclones of PCR product of patient reveals heterozygous A to G transition at position 423 that converts asparagine residue (AAT) to serine residue (AGT). B, AS-PCR analysis of A to G transition at position 423. Genomic DNAs from unrelated individuals and a normal father, mother, sister, and affected son of PC-2 family were analyzed for presence of adenine or guanine at position 423. Only the sample from the affected son showed presence of both adenine (upper column) and guanine (lower column), whereas all other samples showed presence of adenine and absence of guanine.
thought to be derived from a normal allele. With AS-PCR, we confirmed the existence of 7 mutant clones of 21 positive clones. The 267 bp fragment amplified from psoriatic cDNA showed a normal sequence. Mutation analysis Further analysis of the A to G mutation at base pair 423 was undertaken to exclude the possibility that this change occurs in the normal population as a common polymorphism. We used allele-specific primers to assess the presence of this mutation in the DNA of 52 unrelated normal individuals and also in the patient’s parents and sister. The 620-bp fragment was amplified with both the normal allele-specific primer (K17pN) and mutant allelespecific primer (K17pM) in the patient’s DNA. In DNAs from normal individuals, the patient’s parent and sister, the 620-bp fragment was amplified
Journal of the American Academy of Dermatology Volume 38, Number 6, Part 1
Brief communications 1009
only by the normal allele-specific primer (Fig. 2, B).
We thank Mr. Akira Matsumoto for his help with the photography.
DISCUSSION
REFERENCES
In the present study we identified a heterozygous missense mutation within the helix initiation motif of K17 in a Japanese patient with JacksonLawler PC (PC-2). In the type I keratin family, the amino acid sequences of the first 15 codons KVTMQNLNDRLASYL in 1A domain are almost invariant among type I keratins.16,17 A mutation of the K17 gene in a family of PC-2 was initially discovered at position 422 of the K17 genomic sequence, producing an N92D substitution.6 The A to G transition at position 423 in this case occurs at only the base next to the site of the mutation that was determined in a previous report and produces the N92S substitution within the highly conserved helix initiation motif of K17. This mutation has been reported in one familial and three sporadic cases of PC-2.11 Our finding of this mutation in a sporadic case of different ethnic origin supports the thesis that this asparagine (N) codon is a hot spot for the mutation of K17. Recently, similar types of mutation have been reported in two relatives of familial steatocystoma multiplex. The mutations N92H and R94H of K17 were observed in different family pedigrees.11 These cases were not diagnosed as PC and showed only mild deformity of the nails. The reason that similar underlying genetic lesions lead to manifestations of different clinical phenotypes, with or without pachyonychia, is unclear. As previously speculated, each particular mutation of K17 could lead to a phenotypic variation from typical PC2 to familial steatocystoma multiplex with mild or no nail deformity. In this respect, the mutation N92S in the present case is in agreement with the report by Smith et al.11 and supports the clinical diagnosis of typical PC2. Because K17 does not have a specific coexpressed partner keratin, identification of factors that interact with K17 may solve this problem. Elucidation of a number of mutations of the K17 gene will also enhance our understanding of phenotypic heterogeneity of PC and steatocystoma multiplex.
1. Muller C. On the cause of congenital onychogryposis. Muenchen Med Wochenschr 1904;49:2180-2. 2. McKusick VA, editor. Mendelian inheritance in man. 9th ed. Baltimore: The Johns Hopkins University Press; 1990. p. 702-3. 3. Thormann J, Kobayasi T. Pachyonychia congenita Jadasshon-Lewandowsky: a disorder of keratinization. Acta Derm Venereol (Stockh) 1977;57:63-7. 4. Clementi M, Cardin de Stephani E, Dei Rossi C, Avventi V, Tenconi R. Pachyonychia congenita Jackson-Lawler type: a distinct malformation syndrome. Br J Dermatol 1986;114:367-70. 5. Thomas DR, Jorizzo JL, Brysk MM, Tschen JA, Miller J, Tschen EH. Pachyonychia congenita. Arch Dermatol 1984;120:1475-9. 6. McLean WHI, Rugg EL, Lunny DP, Morley SM, Lane EB, Swensson O, et al. Keratin 16 and keratin 17 mutations cause pachyonychia congenita. Nat Genet 1995;9:273-8. 7. Fuchs E. Genetic skin disorders of keratin. J Invest Dermatol 1992;99:671-4. 8. Lane EB. Keratin diseases. Curr Opin Genet Dev 1994;4:412-8. 9. Korge BP, Krieg T. The molecular basis for inherited bullous diseases. J Mol Med 1996;74:59-70. 10. Rothnagel JA. The role of keratin mutations in disorders of the skin. Curr Opin Dermatol 1996;3:127-36. 11. Smith FJD, Corden LD, Rugg EI, Ratnavel R, Leigh IM, Moss C, et al. Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma. J Invest Dermatol 1997;108:220-3. 12. Bowden PE, Harley JL, Kansky A, Rothnagel JA, Jones DO, Turner RJ. Mutation of a type II keratin gene (K6a) in pachyonychia congenita. Nat Genet 1995;10:363-5. 13. Troyanovsky SM, Leube RE, Franke WW. Characterization of the human gene encoding cytokeratin 17 and its expression pattern. Eur J Cell Biol 1992;59:127-37. 14. Tomkova H, Fujimoto W, Arata J. Expression of keratins (K10 and K17) in steatocystoma multiplex, eruptive vellus hair cysts, epidermoid and trichilemmal cysts. Am J Dermatopathol 1997;19:250-3. 15. De Jong EMGJ, Van Vlijmen IMMJ, Van Erp PEJ, Ramaekers FCG, Troyanovski SM, Van de Kerkhof PCM. Keratin 17: a useful marker in anti-psoriatic therapies. Arch Dermatol Res 1991;283:480-2. 16. Conway JF, Parry AD. Intermediate filament structure: 3. Analysis of sequence homologies. Int J Biol Macromol 1988;10:79-98. 17. Hennies H-C, Zehender D, Kunze J, Küster W, Reis A. Keratin 9 gene mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Hum Genet 1994;93:649-54.
Brief communications 1007
Pachyonychia congenita type 2: Keratin 17 mutation in a Japanese case Wataru Fujimoto, MD,a Gen Nakanishi, MD,a Satoshi Hirakawa, MD,a Tohru Nakanishi, PhD,b Tsuyoshi Shimo, DDS,b Masaharu Takigawa, DDS, PhD,b and Jirô Arata, MDa Okayama, Japan Pachyonychia congenita (PC) is an autosomal dominant genodermatosis first described by Muller.1 Two major subtypes of PC have been described.2 Jadassohn-Lewandowsky type PC (MIM #167200; PC1) is characterized by onychogryphosis; hyperkeratosis of the palms, soles, knees, and elbows; follicular hyperkeratosis; and leukoplakia of the oral mucous membranes.3 Jackson-Lawler type PC (MIM #167210; PC2) shows no oral leukoplakia but is characterized by natal teeth and multiple cutaneous cysts and pachyonychia.4 Histopathologic condition of hyperkeratotic skin shows moderate to marked hyperkeratosis and acanthosis. Ultrastructural analysis of keratinocytes in the stratum spinosum of affected areas shows large tonofibril aggregates without signs of cellular disruption or cell death.5,6 The ultrastructural finding of aggregated keratin filament bundles in keratinocytes is a common feature of keratin diseases such as epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic palmoplantar keratoderma, and ichthyosis bullosa of Siemens. In these genetic skin disorders, mutations of keratin genes cause dysfunction of keratin intermediate filaments, leading to blister formation or hyperkeratotic skin lesions.7-10 Heterozygous missense mutations of the keratin 16 gene and keratin 17 (K17) gene have been identified in PC1 and PC2, respectively.6,11 A heterozygous keratin 6a deletion has also been identified in a PC1 family.12 In the present study we report a missense mutation within the helix ini-
From the Department of Dermatology, Okayama University Medical School,a and the Department of Biochemistry and Molecular Dentistry, Okayama University Dental School.b Reprint requests: Wataru Fujimoto, MD, Department of Dermatology, Okayama University Medical School, 2-5-1 Shikata, Okayama 700, Japan. J Am Acad Dermatol 1998;38:1007-9. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/54/89794
tiation motif of the K17 gene in a sporadic Japanese case of Jackson-Lawler type PC. PATIENT AND METHODS
Patient The pedigree examined in this study is shown in Fig. 1, A. The family history of the patient showed no consanguinity and no relatives with similar conditions. The patient is a 24-year-old Japanese man who at birth was covered with white, milia-like cysts (Fig. 1, B) and had two natal teeth. All of his nails were unusually thick from an early age. At 5 or 6 years of age, cysts developed on his face and extremities; the number of cysts gradually increased on his buttocks, groin, and axillae. At 12 or 13 years of age, these cysts became painful, enlarged, and infected. He underwent numerous surgical procedures for infected cysts in the axillae and was diagnosed with hidradenitis suppurativa. Physical examination at the time of the present study revealed numerous cysts and nodules of various sizes all over the body (Fig. 1, C), pachyonychia on all fingers and toes (Fig. 1, D), pili torti (Fig. 1, E), and callus-like focal plantar keratoderma on the left sole. The mouth, tongue, and teeth were normal. Biopsy specimens of several cysts suggested a diagnosis of steatocystoma multiplex. Immunohistochemistry showed positive staining for K17 in the suprabasal lining cells of the cyst wall (Fig. 1, F).
Reverse transcription–polymerase chain reaction and DNA sequencing Because K17 has pseudogenes,13 we performed reverse transcription–polymerase chain reaction to avoid amplifying these genes from genomic DNA. Total RNA was extracted from the patient’s steatocystomas because K17 is abundantly expressed in epithelial cells of the cysts (Fig. 1, F).14 As a control, total RNA was extracted from involved lesions of several patients with psoriasis, because K17 is expressed in abundance in psoriatic lesions.15 The total RNA was reverse transcribed, and the cDNA encoding the entire rod domain of K17 was amplified by PCR with primers k17p3 and k17p6, as described previously,6 and sequenced.
Journal of the American Academy of Dermatology June 1998
1008 Brief communications
A
D
B
C
E
F
Fig. 1. Pedigree of the PC-2 family, clinical photographs of proband and expression of K17 in steatocystoma. A, Family PC-2, sporadic case of JacksonLawler PC. DNA was obtained from four family members (*) including proband (arrow). B, Numerous white, milia-like cysts were seen when the patient was 1 month of age. C, Fresh-colored papules and nodules on anterior chest. D, Thickened fingernails. E, Helical body hair on lower leg. F, Expression of K17 in the lining cells of steatocystoma. Immunohistochemistry was performed on paraffin sections boiled in 10 mmol/L citrate buffer reacted with monoclonal anti-cytokeratin 17, E3 (M7046; Dako, A/S, Denmark).
Allele-specific PCR analysis Allele-specific PCR (AS-PCR) was performed to screen the mutation of subclones containing the 267 base pairs (bp) fragment. The normal allele-specific primer, K17pN (5´-GCCACCATGCAGAACCTCAA3´), and a mutant allele-specific primer, K17pM (5´GCCACCATGCAGAACCTCAG-3´), were used as forward primers, and K17p12 (5´-GCCGGATGTTGGCATTGTC-3´) was used as a common reverse primer. AS-PCR was also performed to screen the mutations in genomic DNAs. K17pN and K17pM were used as the forward primers; K17p10 was used as the reverse primer.6 RESULTS
Sequence analysis of K17 Direct sequencing of amplified 1032 fragments from the patient’s cDNA disclosed an unreadable base in the helix initiation motif of the K17 gene. By subcloning the 267 bp fragment in the helix initiation motif and sequencing some of the positive clones, we identified A to G transition at position 423 in some clones thought to be derived from mutant allele of the patient (Fig. 2, A). This transition results in an asparagine (N) to serine (S) substitution. Other clones showed a normal sequence
A
B Fig. 2. Identification of missense mutation N92S in the K17 gene. A, Nucleotide sequencing of subclones of PCR product of patient reveals heterozygous A to G transition at position 423 that converts asparagine residue (AAT) to serine residue (AGT). B, AS-PCR analysis of A to G transition at position 423. Genomic DNAs from unrelated individuals and a normal father, mother, sister, and affected son of PC-2 family were analyzed for presence of adenine or guanine at position 423. Only the sample from the affected son showed presence of both adenine (upper column) and guanine (lower column), whereas all other samples showed presence of adenine and absence of guanine.
thought to be derived from a normal allele. With AS-PCR, we confirmed the existence of 7 mutant clones of 21 positive clones. The 267 bp fragment amplified from psoriatic cDNA showed a normal sequence. Mutation analysis Further analysis of the A to G mutation at base pair 423 was undertaken to exclude the possibility that this change occurs in the normal population as a common polymorphism. We used allele-specific primers to assess the presence of this mutation in the DNA of 52 unrelated normal individuals and also in the patient’s parents and sister. The 620-bp fragment was amplified with both the normal allele-specific primer (K17pN) and mutant allelespecific primer (K17pM) in the patient’s DNA. In DNAs from normal individuals, the patient’s parent and sister, the 620-bp fragment was amplified
Journal of the American Academy of Dermatology Volume 38, Number 6, Part 1
Brief communications 1009
only by the normal allele-specific primer (Fig. 2, B).
We thank Mr. Akira Matsumoto for his help with the photography.
DISCUSSION
REFERENCES
In the present study we identified a heterozygous missense mutation within the helix initiation motif of K17 in a Japanese patient with JacksonLawler PC (PC-2). In the type I keratin family, the amino acid sequences of the first 15 codons KVTMQNLNDRLASYL in 1A domain are almost invariant among type I keratins.16,17 A mutation of the K17 gene in a family of PC-2 was initially discovered at position 422 of the K17 genomic sequence, producing an N92D substitution.6 The A to G transition at position 423 in this case occurs at only the base next to the site of the mutation that was determined in a previous report and produces the N92S substitution within the highly conserved helix initiation motif of K17. This mutation has been reported in one familial and three sporadic cases of PC-2.11 Our finding of this mutation in a sporadic case of different ethnic origin supports the thesis that this asparagine (N) codon is a hot spot for the mutation of K17. Recently, similar types of mutation have been reported in two relatives of familial steatocystoma multiplex. The mutations N92H and R94H of K17 were observed in different family pedigrees.11 These cases were not diagnosed as PC and showed only mild deformity of the nails. The reason that similar underlying genetic lesions lead to manifestations of different clinical phenotypes, with or without pachyonychia, is unclear. As previously speculated, each particular mutation of K17 could lead to a phenotypic variation from typical PC2 to familial steatocystoma multiplex with mild or no nail deformity. In this respect, the mutation N92S in the present case is in agreement with the report by Smith et al.11 and supports the clinical diagnosis of typical PC2. Because K17 does not have a specific coexpressed partner keratin, identification of factors that interact with K17 may solve this problem. Elucidation of a number of mutations of the K17 gene will also enhance our understanding of phenotypic heterogeneity of PC and steatocystoma multiplex.
1. Muller C. On the cause of congenital onychogryposis. Muenchen Med Wochenschr 1904;49:2180-2. 2. McKusick VA, editor. Mendelian inheritance in man. 9th ed. Baltimore: The Johns Hopkins University Press; 1990. p. 702-3. 3. Thormann J, Kobayasi T. Pachyonychia congenita Jadasshon-Lewandowsky: a disorder of keratinization. Acta Derm Venereol (Stockh) 1977;57:63-7. 4. Clementi M, Cardin de Stephani E, Dei Rossi C, Avventi V, Tenconi R. Pachyonychia congenita Jackson-Lawler type: a distinct malformation syndrome. Br J Dermatol 1986;114:367-70. 5. Thomas DR, Jorizzo JL, Brysk MM, Tschen JA, Miller J, Tschen EH. Pachyonychia congenita. Arch Dermatol 1984;120:1475-9. 6. McLean WHI, Rugg EL, Lunny DP, Morley SM, Lane EB, Swensson O, et al. Keratin 16 and keratin 17 mutations cause pachyonychia congenita. Nat Genet 1995;9:273-8. 7. Fuchs E. Genetic skin disorders of keratin. J Invest Dermatol 1992;99:671-4. 8. Lane EB. Keratin diseases. Curr Opin Genet Dev 1994;4:412-8. 9. Korge BP, Krieg T. The molecular basis for inherited bullous diseases. J Mol Med 1996;74:59-70. 10. Rothnagel JA. The role of keratin mutations in disorders of the skin. Curr Opin Dermatol 1996;3:127-36. 11. Smith FJD, Corden LD, Rugg EI, Ratnavel R, Leigh IM, Moss C, et al. Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma. J Invest Dermatol 1997;108:220-3. 12. Bowden PE, Harley JL, Kansky A, Rothnagel JA, Jones DO, Turner RJ. Mutation of a type II keratin gene (K6a) in pachyonychia congenita. Nat Genet 1995;10:363-5. 13. Troyanovsky SM, Leube RE, Franke WW. Characterization of the human gene encoding cytokeratin 17 and its expression pattern. Eur J Cell Biol 1992;59:127-37. 14. Tomkova H, Fujimoto W, Arata J. Expression of keratins (K10 and K17) in steatocystoma multiplex, eruptive vellus hair cysts, epidermoid and trichilemmal cysts. Am J Dermatopathol 1997;19:250-3. 15. De Jong EMGJ, Van Vlijmen IMMJ, Van Erp PEJ, Ramaekers FCG, Troyanovski SM, Van de Kerkhof PCM. Keratin 17: a useful marker in anti-psoriatic therapies. Arch Dermatol Res 1991;283:480-2. 16. Conway JF, Parry AD. Intermediate filament structure: 3. Analysis of sequence homologies. Int J Biol Macromol 1988;10:79-98. 17. Hennies H-C, Zehender D, Kunze J, Küster W, Reis A. Keratin 9 gene mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Hum Genet 1994;93:649-54.