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Cockayne syndrome without typical clinical manifestations including neurologic abnormalities
Cockayne syndrome without typical clinical manifestations including neurologic abnormalities Hiroko Miyauchi-Hashimoto, MD,a Tamiyo Akaeda, MD,a Toshiro Maihara, MD,b Mituo Ikenaga, PhD,c and Takeshi Horio, MDa Moriguchi and Kyoto, Japan Although patients with mild symptoms of atypical Cockayne syndrome (CS) have been described, there has not been a report of a patient with CS whose only clinical manifestation was cutaneous photosensitivity. Cells from patients with CS show UV sensitivity, reduced recovery of RNA synthesis, but normal UV-induced unscheduled DNA synthesis. On the other hand, the patients with UV-sensitive syndrome have only cutaneous photosensitivity and skin freckles, whereas those cells respond to UV radiation in a similar fashion to the CS cells. We describe a patient with CS who showed only photosensitivity without typical clinical manifestations of CS, but his cells showed UV sensitivity, reduced recovery of RNA synthesis, and normal unscheduled DNA synthesis after UV radiation similar to CS cells. Furthermore, the patient was assigned to complementation group B of CS on the basis of the results of complementation analysis. The present report suggests that CS has a wider spectrum than that considered previously. (J Am Acad Dermatol 1998;39:565-70.)
Cockayne syndrome (CS) is a rare autosomal recessive disorder characterized by various clinical manifestations, such as cachectic dwarfism, deafness, retinal pigmentation, cutaneous photosensitivity, a thickened skull, intracranial calcification, mental deficiency, and characteristic facial features.1,2 However, patients with CS do not have actinically induced skin cancer,3,4 unlike those with xeroderma pigmentosum (XP), which is also one of the genodermatoses characterized by photosensitivity. Cultured cells from patients with CS show highly increased sensitivity to the lethal effects of UV radiation5 similar to that in XP, but CS cells have a normal level of UV-induced unscheduled DNA synthesis (UDS) unlike XP cells. In CS cells the recovery of RNA synthesis after UV irradiation does not occur or is markedly delayed,6 whereas in normal cells it is only temporarily depressed. Recent studies have demonstrated that this phenomenon was due to defective From the Department of Dermatology, Kansai Medical University,a Department of Pediatrics, Faculty of Medicine,b and the Radiation Biology Center,c Kyoto University. Accepted for publication June 26, 1998. Reprint requests: Hiroko Miyauchi-Hashimoto, MD, Department of Dermatology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka, 570-8507, Japan. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/1/92717
preferential repair of DNA damage in actively transcribing genes.7,8 Two complementation groups of CS (groups A and B) have been identified. CS is a variable disorder with a great range in type and severity of signs and symptoms. It may be difficult to define the syndrome clearly, and several atypical cases have been described.4,9-14 Previously described patients with only mild symptoms of CS had some neurologic problems or growth retardation with or without photosensitivity. Itoh et al15,16 described 2 patients having no clinical manifestation except for photosensitivity and cutaneous pigmentation, similar to a mild XP phenotype. Their cellular characteristics were the same as CS, such as UV hypersensitivity, defective recovery of RNA synthesis after UV radiation, and normal levels of UDS. However, the result of complementation analysis showed that these patients do not belong to any of the known complementation groups of CS or XP. Hence these patients were classified as having “UV-sensitive” (UVs) syndrome, a new category of photosensitive disease. We present herein an atypical patient with CS whose only clinical manifestations were photosensitivity and pigmented freckles on sun-exposed areas, similar to those seen in a person with mild XP phenotype or UVs syndrome. Cultured skin 565
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ines) were present. He was 114.5 cm tall, weighed 22 kg, and his head circumference was 52 cm (normal values of a 5-year-old Japanese boy: 110.49 ± 4.51 cm, 19.07 ± 2.84 kg, and 51.0 ± 2.1 cm, respectively). Up to the time of this report, he had no obvious growth retardation, microcephaly, senile appearance, or other congenital malformations. His intelligence quotient was 96. Neurologic examination did not reveal ataxia or tremor; muscle tonus and tendon reflex were normal. Ophthalmologic examination and audiogram did not reveal any abnormalities. The results of laboratory studies were normal. The findings of a computed tomographic scan and roentgenography disclosed neither brain atrophy nor calcification. The patient was observed until he was 13 years old. At that time, he was a well-developed athlete and his school record was good. He has been normal except for photosensitivity; results of other examinations have not shown abnormal findings. MATERIAL AND METHODS
Fig 1. Erythema and pigmented freckles on the nose and cheeks.
fibroblasts of the patient showed extreme UV sensitivity and defect of recovery of RNA synthesis after UV radiation, but had an almost normal amount of UDS. The clinical features and cellular characteristics of our patient were similar to UVs syndrome. However, the patient was assigned to group B of CS by complementation analysis, which measures the recovery of RNA synthesis after UV in fused binuclear cells. This suggests that UVs syndrome is closely akin to CS or may be the third complementation group of CS. CASE REPORT A 13-year-old Japanese boy was first seen in our University Hospital at 5 years of age because of severe recurrent sunburns. His prenatal, natal, and postnatal histories were normal. He had shown photosensitivity at 4 months of age. He matured normally except for an increased risk of sunburn after long sun exposure. His parents are not consanguineous and the patient has 1 younger sibling. His parents, younger sibling, and relatives showed neither abnormal sun sensitivity nor clinical manifestations of CS. At the first visit, he showed edematous erythema on the cheek, nose, and nape (Fig 1). The pigmented freckling and scaling were scattered over his face. On other sites no skin lesions (eg, xerosis, anhidrosis, or lentig-
Cells. The primary skin fibroblast cell culture (CS3AM) derived from the patient, 2 normal cell strains (CHFU17 and N17OS17), and 4 established CS cell lines (GM1856, GM1098, CS1OS, and CS1MO) were used. The strains GM185618 and CS1OS19,20 belong to complementation group A. The strains GM109818 and CS1MO15,21 belong to complementation group B. The CHFU, N17OS, CS1OS, and CS1MO strains were developed in our laboratory and strains GM1098 and GM1856 were purchased from Human Genetic Mutant Cell Repository, Camden, New Jersey. All cell strains were grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with 10% fetal calf serum (Hyclone Laboratory, Logan, Utah) at 37°C in a humidified atmosphere containing 10% CO2. CS3AM cells were used between the third and fifth passages. UV sensitivity of cells. UV sensitivity test by colony-forming assay was performed as previously described.14,17 Appropriate numbers of cells were seeded in 60-mm dishes. After 14 to 16 hours’ incubation, cells were washed with phosphate-buffered saline (PBS) and irradiated with UVC at a fluence rate of 1.4 W/m2. The light source was a bank of 2 germicidal lamps (Hitachi GL15, Hitachi, Tokyo, Japan) emitting predominantly at a wavelength of 254 nm. The UVCirradiated cells were incubated for 14 to 16 days with medium renewal twice a week, and colonies were counted after staining. Measurement of unscheduled DNA synthesis after UV irradiation. The measurement method used was described previously.14,17 Briefly, cells were seeded on cover slips and incubated for 24 hours. The cells were washed with PBS, irradiated with UVC at a dose
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of 30 J/m2, labeled for 3 hours in a medium containing 10 µCi/mL of [methyl-3H]thymidine (25 Ci/mmol; Amersham International, Buckinghamshire, UK), and further incubated for l hour in medium containing 5 µg/mL of nonradioactive thymidine. Autoradiography was performed with Konica NR-M2 emulsion (Konica Co, Tokyo, Japan). After exposure for 2 weeks at 4°C the number of grains per nucleus was counted in Giemsa-stained cells. The mean number of grains was determined by counting 100 nuclei per specimen. Genetic complementation test. Complementation analysis was performed as previously described.14,18 The 2 different CS cell strains to be fused were mixed with an equal amount (5 × 104 cells each), seeded on coverslips and incubated for 24 hours. The cells were fused by treatment with 45% polyethylene glycol (PEG#6000, Nakalai Tesque, Kyoto, Japan) for 1 minute, washed, and incubated for 3 days in DMEM containing 0.5% FCS to arrest cell growth. Subsequently, the cells were washed, irradiated with UVC at a dose of 20 J/cm2, incubated in medium containing 0.5% FCS for 24 hours, and labeled for l hour in a medium containing 15 µCi/mL of tritiated uridine (25 Ci/mmol; Amersham International). Autoradiography was carried out with an exposure time of 3 days, and the cells were then stained with Giemsa. The number of grains per nucleus was counted in more than 50 nuclei each of mononuclear and binuclear cells. Phototesting. Photosensitivity tests were performed on the back of the patient with a bank of 7 fluorescent sunlamps (Toshiba FL20SE30, Toshiba Medical Supply, Tokyo, Japan) that emits light of 280 to 370 nm (mainly UVB, peaking at 305 nm) and a bank of 14 fluorescent black lights (Toshiba FL32S.BL, Toshiba Medical Supply) that emits rays between 300 and 430 nm, with a peak at 352 nm. RESULTS
Survival after UV irradiation. Fig 2 shows the survival of the UV-irradiated patient’s cells, as measured by colony-forming ability in comparison with the normal cell strain (CHFU). Each UV survival was determined by 3 independent examinations. Our patient’s strain (CS3AM) was significantly more UV sensitive than normal cells. In addition, the survival curve of our patient’s strain was similar to those of the CS1OS (group A) and CS1MO (group B) strains from the patients with typical manifestations of CS. UDS. To know the DNA repair capacity of CS3AM cells relative to normal (CHFU) cells, we measured the amounts of UDS after UV irradiation by autoradiography. The average number of grains per nucleus of CS3AM and CHFU cells
Miyauchi-Hashimoto et al 567
Fig 2. UV survivals of CS cells. Colony-forming abilities of fibroblasts after irradiation with different doses of UVC. ▲ , CHFU (normal); d, CS3AM (present case); h, CS1MO (group B CS) (classic CS); s, CS1OS (group A CS).
were 96.74 and 103.36, respectively. Thus the UDS level in CS3AM cell was 94% of normal cells. Complementation analysis. We performed complementation tests by the method in which recovery of RNA synthesis after UV radiation was examined in fused cells. The representative data are shown in Fig 3. The histogram shows the distribution of the number of grains per nucleus in fused binuclear cells. As a positive control, the GM1856 (group A) strain was fused with the GM1098 (group B) strain. When fused with themselves, GM1856 or GM1098 cells have a low average grain number (8.52 ± 4.23 or 22.56 ± 7.07, respectively). Because these 2 cell strains belong to different complementation groups, the average nuclear grain count in binuclear cells fused between GM1856 and GM1098 increased (60.68 ± 26.25, Fig 3, d), nearly to the levels of normal CHFU cells (74.08 ± 19.19). Whereas the CS3AM
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Fig 3. Complementation analysis of CS3AM. CS3AM cells were hybridized with (a) itself, (b) GM1856 (group A), and (c) GM1098 (group B). As a positive control, GM1856 cells were fused with GM1098 (d). Histogram shows distributions of the number of grains per nucleus scored only with binuclear cells. Numbers indicated with arrows represent average number of grains.
strain fused with itself has a low average grain count (8.52 ± 5.11, Fig 3, A), the average grain count in binuclear cells of CS3AM fused with GM1856 increased (61.46 ± 43.8, Fig 3, B), indicating that these 2 strains belong to the different complementation groups. On the other hand, binuclear cells of CS3AM fused with GM1098 have a low average grain count (9.86 ± 5.75, Fig 3, C), indicating that they belong to the same complementation group. Similarly, the CS3AM cell complemented the group A CS1OS cell line but not the group B CS1MO cell line (data not shown). Therefore the CS3AM strain was unambiguously assigned to group B. Phototesting. The minimal erythema dose (MED) of UVB, which was determined 24 hours after exposure, was 34 mJ/cm2 in our patient. This was significantly lower than the mean value (90 mJ/cm2) for healthy Japanese subjects.22 Although the reaction did not become more marked, almost the same intensity of erythema and edema remained 48 hours after irradiation. No erythema developed at the site exposed to UVA at a dose as high as 48 J/cm2. DISCUSSION
We described an unusual patient with CS whose clinical manifestation was restricted to the skin
photosensitivity. This suggests that CS has a wider spectrum than considered previously. In CS, affected children are products of a normal pregnancy and appear normal at birth. Childhood is normal until mental retardation and growth retardation are discovered between 6 months and 4 years of age.4,23 Subsequently the full syndrome develops. Many neurologic symptoms are progressive, and most patients die before reaching 20 years of age. Nance and Berry4 reviewed 140 published cases of CS and suggested that, for classic CS, the clinical diagnosis requires growth failure and neurodevelopmental delay together with 3 of the following: retinopathy/cataracts, hearing loss, dental caries, photosensitivity, and characteristic facial appearance. Because CS is diagnosed from the clinical manifestations characterized by a great variety of symptoms, our patient was not immediately diagnosed with CS. Nance and Berry classified CS into 3 clinical subtypes: classic CS, severe CS, and mild CS. The patients classified into the “mild CS” group whose symptoms are partial, mild, or late onset have some neurologic findings and/or growth retardation. Thus the patient described herein is atypical even for a case of “mild CS.” Cultured fibroblasts from our patient had marked UV sensitivity and reduced recovery of RNA syn-
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thesis after UV radiation, but had a normal level of UDS, consistent with characteristics of CS cells. Furthermore, the complementation analysis demonstrated that the defect in our patient’s cells belong to complementation group B of CS. To our knowledge, ours is the first report of a patient with CS who was assigned to a specific CS complementation group, despite the absence of neurologic abnormalities. Cellular studies on fibroblasts are useful to make an accurate diagnosis of CS. Clinical photosensitivity is one of the characteristic features of this syndrome. Although photosensitivity was present in approximately three fourths of affected persons,4 in vivo phototests were performed only in limited cases. Some patients with CS have shown an abnormal delayed reaction or lowered MED to UVB irradiation. Our patient also had abnormal responses to UVB, such as reduction of MED and longer-lasting erythema and edema, but had normal reactivity to UVA. However, it is not clear that such photosensitivity is common in all patients with CS because accumulated data are insufficient. Lehmann et al24 reported that the failure of RNA synthesis to recover after UV irradiation provides a useful and relatively simple diagnostic test for CS. Along this line, Cleaver and Thomas25 described some cases who showed photosensitivity as the sole clinical manifestation, whereas their cellular responses were similar to CS, namely, defective recovery of RNA synthesis and hypersensitivity to killing by UV. Recently, Itoh, Ono, and Yamaizumi15 have described 2 Japanese siblings who were photosensitive without other clinical manifestations of CS, even though their skin fibroblasts showed UV responses characteristic of CS. Furthermore, they assigned these patients and the patient described by Fujiwara et al26 (who is one of the patients reviewed by Cleaver and Thomas25), by complementation analysis, to the same but a new complementation group termed UVs syndrome, which neither belongs to any known groups of CS (groups A and B) nor XP (A through G).15,16 There are no differences between the present case and UVs syndrome in the clinical and cellular characteristics, although they are genetically different. Therefore our patient provides a possibility that UVs syndrome may be a new complementation group of CS, or, at least, a closely related disease. The wide spectrum of clinical manifestations of
Miyauchi-Hashimoto et al 569 CS suggests that biologic and genetic heterogeneity exists. The CS-B gene (also called the ERCC6 gene) has been cloned and characterized.27,28 This revealed that it is a relatively large gene with at least 21 exons and encodes a protein of 168 kd, which contains a DNA helicase motif. Because CS-B cells are defective in the preferential repair of template (transcribed) strands in the actively transcribed genes,7,27,28 the CS-B protein is thought to be involved in the coupling of RNA transcription to excision repair of UV damage. The clinical diversity in CS-B may be due to different mutations occurring at different sites within the CS-B gene, as suggested by the mutational analysis with various group A XP cells.29,30 The present patient without neurologic abnormalities may have a particular mutation in the CS-B gene, distinctly different from mutations detectable in typical CS-B patients. Alternatively, in the present case a mutation could be induced at a certain stage of embryonic development; thus the CS phenotype would be expressed only in restricted organs, such as the skin. We previously reported an evidence of such somatic mosaicism for an XP-A patient.17 In any case, analysis of the mutation in our patient’s cells will solve this problem. REFERENCES 1. Cockayne EA. Dwarfism with retinal atrophy and deafness. Arch Dis Child 1936;11:1-8. 2. Summitt RL. Cockayne syndrome. In: Bergsma D, editor. Birth defect compendium. 2nd ed. New York: Alan R. Liss; 1979. p. 236-7. 3. Lehmann AR. Cockayne’s syndrome and trichothiodystrophy: defective repair without cancer. Cancer Rev 1987;7:82-103. 4. Nance MA, Berry SA. Cockayne syndrome: review of 140 cases. Am J Med Genet 1992;42:68-84. 5. Andrews AD, Barrett SF, Yoder FW, Robbins JH. Cockayne’s syndrome fibroblasts have increased sensitivity to ultraviolet light but normal rates of unscheduled DNA synthesis. J Invest Dermatol 1978;70:237-9. 6. Mayne LV, Lehmann AR. Failure of RNA synthesis to recover after UV-irradiation: an early defect in cells from individuals with Cockayne’s syndrome and xeroderma pigmentosum. Cancer Res 1982;42:1473-8. 7. van Hoffen A, Natarajan AT, Mayne LV, van Zeeland AA, Mullenders LH, Venema J. Deficient repair of the transcribed strand of active genes in Cockayne’s syndrome cells. Nucleic Acids Res 1993;21:5890-5. 8. Barrett SF, Robbins JH, Tarone RE, Kraemer KH. Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells. Mutat Res 1991;255:281-91. 9. Lanning M, Simila S. Cockayne’s syndrome: report of a
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10. 11.
12. 13.
14. 15.
16.
17.
18. 19.
20.
case with normal intelligence. Z Kinderheilk 1970;109: 70-5. Soffer D, Grotsky HW, Rapin I, Suzuki K. Cockayne syndrome: unusual neuropathological findings and review of the literature. Ann Neurol 1979;6:340-8. Ohta S, Shima A, Shimada M, Katsura T, Nozue T, Ohta S. Ultraviolet sensitivity of Cockayne syndrome and its heterozygotes: cell biological diagnosis of atypical case. Cong Anom 1983;23:399-403. Touge T, Takeuchi H, Miki H, Yamada A, Nishioka M. A case of Cockayne syndrome and analysis of 50 cases in Japan. Intern Med 1986;58:1058-61. (In Japanese) Inoue K, Hanyu N, Takatsu M, Yanagisawa N, Tsukagoshi H. The Cockayne’s syndrome with benign clinical course: a study of four cases from two families. Clin Neurol 1978;18:477-85. (In Japanese) Miyauchi H, Horio T, Akaeda T, Asada A, Chang HR, Ishizaki K, et al. Cockayne syndrome in two adult siblings. J Am Acad Dermatol 1994;30:329-35. Itoh T, Ono T, Yamaizumi M. A new UV-sensitive syndrome not belonging to any complementation groups of xeroderma pigmentosum or Cockayne syndrome: siblings showing biochemical characteristics of Cockayne syndrome without typical clinical manifestations. Mutat Res 1994;314:233-48. Itoh T, Fujiwara Y, Ono T, Yamaizumi M. UVS syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group I. Am J Hum Genet 1995;56:1267-76. Chang HR, Ishizaki K, Sasaki M, et al. Somatic mosaicism for DNA repair capacity in fibroblasts derived from a group A xeroderma pigmentosum patient. J Invest Dermatol 1989;93:460-5. Lehmann AR. Three complementation groups in Cockayne syndrome. Mutat Res 1982;106:347-56. Sugita T, Ikenaga M, Suehara N, Kozuka T, Furuyama J, Yabuuchi H. Prenatal diagnosis of Cockayne syndrome using assay of colony-forming ability in ultraviolet light irradiated cells. Clin Genet 1982;22:137-42. Ayaki H, Hara R, Ikenaga M. Recovery from ultraviolet light-induced depression of ribosomal RNA synthesis in
21. 22. 23.
24.
25. 26.
27.
28.
29.
30.
normal human, xeroderma pigmentosum and Cockayne syndrome cells. J Radiat Res 1996;37:107-16. Noda A, Ikenaga M. Genetic complementation groups of Cockayne syndrome in Japan [abstract]. J Radiat Res 1985;26:52. Horio T. Actinic reticuloid via persistent light reaction from photoallergic contact dermatitis. Arch Dermatol 1982;118:339-42. Brumback RA, Yoder FW, Andrews AD, Peck GL, Robbins JH. Normal pressure hydrocephalus: recognition and relationship to neurological abnormalities in Cockayne’s syndrome. Arch Neurol 1978;35:337-45. Lehmann AR, Thompson AF, Harcourt SA, Stefanini M, Norris PG. Cockayne’s syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation. J Med Genet 1993;30:679-82. Cleaver JE, Thomas GH. Clinical syndromes associated with DNA repair deficiency and enhanced sun sensitivity. Arch Dermatol 1993;129:348-50. Fujiwara Y, Ichihashi M, Kano Y, Goto K, Shimizu K. A new human photosensitive subject with a defect in the recovery of DNA synthesis after ultraviolet-light irradiation. J Invest Dermatol 1981;77:256-63. Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D, Hoeijmakers JHJ. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s syndrome and preferential repair of active genes. Cell 1992;71:939-53. Troelstra C, Hesen W, Bootsma D, Hoeijmakers JHJ. Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne’s syndrome group B. Nucl Acids Res 1993;21:419-26. Satokata I, Tanaka K, Yuba S, Okada Y. Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A xeroderma pigmentosum. Mutat Res 1992;273:203-12. Nishigori C, Moriwaki S, Takebe H, Tanaka T, Imamura S. Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan. Arch Dermatol 1994;130:191-7.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of the Journal of the American Academy of Dermatology for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted at a cost of $16.00 per issue. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one third off on quantities of 24 or more. Please write to Mosby, Inc., Periodical Subscription Services, 11830 Westline Industrial Dr., St. Louis, MO 63146-3318, or call (800)453-4351 or (314)453-4351 for information on availability of particular issues. If unavailable from the publisher, photocopies of complete issues may be purchased from UMI, 300 N. Zeeb Rd., Ann Arbor, MI 48106, (313)761-4700.
Cockayne syndrome (CS) is a rare autosomal recessive disorder characterized by various clinical manifestations, such as cachectic dwarfism, deafness, retinal pigmentation, cutaneous photosensitivity, a thickened skull, intracranial calcification, mental deficiency, and characteristic facial features.1,2 However, patients with CS do not have actinically induced skin cancer,3,4 unlike those with xeroderma pigmentosum (XP), which is also one of the genodermatoses characterized by photosensitivity. Cultured cells from patients with CS show highly increased sensitivity to the lethal effects of UV radiation5 similar to that in XP, but CS cells have a normal level of UV-induced unscheduled DNA synthesis (UDS) unlike XP cells. In CS cells the recovery of RNA synthesis after UV irradiation does not occur or is markedly delayed,6 whereas in normal cells it is only temporarily depressed. Recent studies have demonstrated that this phenomenon was due to defective From the Department of Dermatology, Kansai Medical University,a Department of Pediatrics, Faculty of Medicine,b and the Radiation Biology Center,c Kyoto University. Accepted for publication June 26, 1998. Reprint requests: Hiroko Miyauchi-Hashimoto, MD, Department of Dermatology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka, 570-8507, Japan. Copyright © 1998 by the American Academy of Dermatology, Inc. 0190-9622/98/$5.00 + 0 16/1/92717
preferential repair of DNA damage in actively transcribing genes.7,8 Two complementation groups of CS (groups A and B) have been identified. CS is a variable disorder with a great range in type and severity of signs and symptoms. It may be difficult to define the syndrome clearly, and several atypical cases have been described.4,9-14 Previously described patients with only mild symptoms of CS had some neurologic problems or growth retardation with or without photosensitivity. Itoh et al15,16 described 2 patients having no clinical manifestation except for photosensitivity and cutaneous pigmentation, similar to a mild XP phenotype. Their cellular characteristics were the same as CS, such as UV hypersensitivity, defective recovery of RNA synthesis after UV radiation, and normal levels of UDS. However, the result of complementation analysis showed that these patients do not belong to any of the known complementation groups of CS or XP. Hence these patients were classified as having “UV-sensitive” (UVs) syndrome, a new category of photosensitive disease. We present herein an atypical patient with CS whose only clinical manifestations were photosensitivity and pigmented freckles on sun-exposed areas, similar to those seen in a person with mild XP phenotype or UVs syndrome. Cultured skin 565
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ines) were present. He was 114.5 cm tall, weighed 22 kg, and his head circumference was 52 cm (normal values of a 5-year-old Japanese boy: 110.49 ± 4.51 cm, 19.07 ± 2.84 kg, and 51.0 ± 2.1 cm, respectively). Up to the time of this report, he had no obvious growth retardation, microcephaly, senile appearance, or other congenital malformations. His intelligence quotient was 96. Neurologic examination did not reveal ataxia or tremor; muscle tonus and tendon reflex were normal. Ophthalmologic examination and audiogram did not reveal any abnormalities. The results of laboratory studies were normal. The findings of a computed tomographic scan and roentgenography disclosed neither brain atrophy nor calcification. The patient was observed until he was 13 years old. At that time, he was a well-developed athlete and his school record was good. He has been normal except for photosensitivity; results of other examinations have not shown abnormal findings. MATERIAL AND METHODS
Fig 1. Erythema and pigmented freckles on the nose and cheeks.
fibroblasts of the patient showed extreme UV sensitivity and defect of recovery of RNA synthesis after UV radiation, but had an almost normal amount of UDS. The clinical features and cellular characteristics of our patient were similar to UVs syndrome. However, the patient was assigned to group B of CS by complementation analysis, which measures the recovery of RNA synthesis after UV in fused binuclear cells. This suggests that UVs syndrome is closely akin to CS or may be the third complementation group of CS. CASE REPORT A 13-year-old Japanese boy was first seen in our University Hospital at 5 years of age because of severe recurrent sunburns. His prenatal, natal, and postnatal histories were normal. He had shown photosensitivity at 4 months of age. He matured normally except for an increased risk of sunburn after long sun exposure. His parents are not consanguineous and the patient has 1 younger sibling. His parents, younger sibling, and relatives showed neither abnormal sun sensitivity nor clinical manifestations of CS. At the first visit, he showed edematous erythema on the cheek, nose, and nape (Fig 1). The pigmented freckling and scaling were scattered over his face. On other sites no skin lesions (eg, xerosis, anhidrosis, or lentig-
Cells. The primary skin fibroblast cell culture (CS3AM) derived from the patient, 2 normal cell strains (CHFU17 and N17OS17), and 4 established CS cell lines (GM1856, GM1098, CS1OS, and CS1MO) were used. The strains GM185618 and CS1OS19,20 belong to complementation group A. The strains GM109818 and CS1MO15,21 belong to complementation group B. The CHFU, N17OS, CS1OS, and CS1MO strains were developed in our laboratory and strains GM1098 and GM1856 were purchased from Human Genetic Mutant Cell Repository, Camden, New Jersey. All cell strains were grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with 10% fetal calf serum (Hyclone Laboratory, Logan, Utah) at 37°C in a humidified atmosphere containing 10% CO2. CS3AM cells were used between the third and fifth passages. UV sensitivity of cells. UV sensitivity test by colony-forming assay was performed as previously described.14,17 Appropriate numbers of cells were seeded in 60-mm dishes. After 14 to 16 hours’ incubation, cells were washed with phosphate-buffered saline (PBS) and irradiated with UVC at a fluence rate of 1.4 W/m2. The light source was a bank of 2 germicidal lamps (Hitachi GL15, Hitachi, Tokyo, Japan) emitting predominantly at a wavelength of 254 nm. The UVCirradiated cells were incubated for 14 to 16 days with medium renewal twice a week, and colonies were counted after staining. Measurement of unscheduled DNA synthesis after UV irradiation. The measurement method used was described previously.14,17 Briefly, cells were seeded on cover slips and incubated for 24 hours. The cells were washed with PBS, irradiated with UVC at a dose
Journal of the American Academy of Dermatology Volume 39, Number 4, Part 1
of 30 J/m2, labeled for 3 hours in a medium containing 10 µCi/mL of [methyl-3H]thymidine (25 Ci/mmol; Amersham International, Buckinghamshire, UK), and further incubated for l hour in medium containing 5 µg/mL of nonradioactive thymidine. Autoradiography was performed with Konica NR-M2 emulsion (Konica Co, Tokyo, Japan). After exposure for 2 weeks at 4°C the number of grains per nucleus was counted in Giemsa-stained cells. The mean number of grains was determined by counting 100 nuclei per specimen. Genetic complementation test. Complementation analysis was performed as previously described.14,18 The 2 different CS cell strains to be fused were mixed with an equal amount (5 × 104 cells each), seeded on coverslips and incubated for 24 hours. The cells were fused by treatment with 45% polyethylene glycol (PEG#6000, Nakalai Tesque, Kyoto, Japan) for 1 minute, washed, and incubated for 3 days in DMEM containing 0.5% FCS to arrest cell growth. Subsequently, the cells were washed, irradiated with UVC at a dose of 20 J/cm2, incubated in medium containing 0.5% FCS for 24 hours, and labeled for l hour in a medium containing 15 µCi/mL of tritiated uridine (25 Ci/mmol; Amersham International). Autoradiography was carried out with an exposure time of 3 days, and the cells were then stained with Giemsa. The number of grains per nucleus was counted in more than 50 nuclei each of mononuclear and binuclear cells. Phototesting. Photosensitivity tests were performed on the back of the patient with a bank of 7 fluorescent sunlamps (Toshiba FL20SE30, Toshiba Medical Supply, Tokyo, Japan) that emits light of 280 to 370 nm (mainly UVB, peaking at 305 nm) and a bank of 14 fluorescent black lights (Toshiba FL32S.BL, Toshiba Medical Supply) that emits rays between 300 and 430 nm, with a peak at 352 nm. RESULTS
Survival after UV irradiation. Fig 2 shows the survival of the UV-irradiated patient’s cells, as measured by colony-forming ability in comparison with the normal cell strain (CHFU). Each UV survival was determined by 3 independent examinations. Our patient’s strain (CS3AM) was significantly more UV sensitive than normal cells. In addition, the survival curve of our patient’s strain was similar to those of the CS1OS (group A) and CS1MO (group B) strains from the patients with typical manifestations of CS. UDS. To know the DNA repair capacity of CS3AM cells relative to normal (CHFU) cells, we measured the amounts of UDS after UV irradiation by autoradiography. The average number of grains per nucleus of CS3AM and CHFU cells
Miyauchi-Hashimoto et al 567
Fig 2. UV survivals of CS cells. Colony-forming abilities of fibroblasts after irradiation with different doses of UVC. ▲ , CHFU (normal); d, CS3AM (present case); h, CS1MO (group B CS) (classic CS); s, CS1OS (group A CS).
were 96.74 and 103.36, respectively. Thus the UDS level in CS3AM cell was 94% of normal cells. Complementation analysis. We performed complementation tests by the method in which recovery of RNA synthesis after UV radiation was examined in fused cells. The representative data are shown in Fig 3. The histogram shows the distribution of the number of grains per nucleus in fused binuclear cells. As a positive control, the GM1856 (group A) strain was fused with the GM1098 (group B) strain. When fused with themselves, GM1856 or GM1098 cells have a low average grain number (8.52 ± 4.23 or 22.56 ± 7.07, respectively). Because these 2 cell strains belong to different complementation groups, the average nuclear grain count in binuclear cells fused between GM1856 and GM1098 increased (60.68 ± 26.25, Fig 3, d), nearly to the levels of normal CHFU cells (74.08 ± 19.19). Whereas the CS3AM
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Fig 3. Complementation analysis of CS3AM. CS3AM cells were hybridized with (a) itself, (b) GM1856 (group A), and (c) GM1098 (group B). As a positive control, GM1856 cells were fused with GM1098 (d). Histogram shows distributions of the number of grains per nucleus scored only with binuclear cells. Numbers indicated with arrows represent average number of grains.
strain fused with itself has a low average grain count (8.52 ± 5.11, Fig 3, A), the average grain count in binuclear cells of CS3AM fused with GM1856 increased (61.46 ± 43.8, Fig 3, B), indicating that these 2 strains belong to the different complementation groups. On the other hand, binuclear cells of CS3AM fused with GM1098 have a low average grain count (9.86 ± 5.75, Fig 3, C), indicating that they belong to the same complementation group. Similarly, the CS3AM cell complemented the group A CS1OS cell line but not the group B CS1MO cell line (data not shown). Therefore the CS3AM strain was unambiguously assigned to group B. Phototesting. The minimal erythema dose (MED) of UVB, which was determined 24 hours after exposure, was 34 mJ/cm2 in our patient. This was significantly lower than the mean value (90 mJ/cm2) for healthy Japanese subjects.22 Although the reaction did not become more marked, almost the same intensity of erythema and edema remained 48 hours after irradiation. No erythema developed at the site exposed to UVA at a dose as high as 48 J/cm2. DISCUSSION
We described an unusual patient with CS whose clinical manifestation was restricted to the skin
photosensitivity. This suggests that CS has a wider spectrum than considered previously. In CS, affected children are products of a normal pregnancy and appear normal at birth. Childhood is normal until mental retardation and growth retardation are discovered between 6 months and 4 years of age.4,23 Subsequently the full syndrome develops. Many neurologic symptoms are progressive, and most patients die before reaching 20 years of age. Nance and Berry4 reviewed 140 published cases of CS and suggested that, for classic CS, the clinical diagnosis requires growth failure and neurodevelopmental delay together with 3 of the following: retinopathy/cataracts, hearing loss, dental caries, photosensitivity, and characteristic facial appearance. Because CS is diagnosed from the clinical manifestations characterized by a great variety of symptoms, our patient was not immediately diagnosed with CS. Nance and Berry classified CS into 3 clinical subtypes: classic CS, severe CS, and mild CS. The patients classified into the “mild CS” group whose symptoms are partial, mild, or late onset have some neurologic findings and/or growth retardation. Thus the patient described herein is atypical even for a case of “mild CS.” Cultured fibroblasts from our patient had marked UV sensitivity and reduced recovery of RNA syn-
Journal of the American Academy of Dermatology Volume 39, Number 4, Part 1
thesis after UV radiation, but had a normal level of UDS, consistent with characteristics of CS cells. Furthermore, the complementation analysis demonstrated that the defect in our patient’s cells belong to complementation group B of CS. To our knowledge, ours is the first report of a patient with CS who was assigned to a specific CS complementation group, despite the absence of neurologic abnormalities. Cellular studies on fibroblasts are useful to make an accurate diagnosis of CS. Clinical photosensitivity is one of the characteristic features of this syndrome. Although photosensitivity was present in approximately three fourths of affected persons,4 in vivo phototests were performed only in limited cases. Some patients with CS have shown an abnormal delayed reaction or lowered MED to UVB irradiation. Our patient also had abnormal responses to UVB, such as reduction of MED and longer-lasting erythema and edema, but had normal reactivity to UVA. However, it is not clear that such photosensitivity is common in all patients with CS because accumulated data are insufficient. Lehmann et al24 reported that the failure of RNA synthesis to recover after UV irradiation provides a useful and relatively simple diagnostic test for CS. Along this line, Cleaver and Thomas25 described some cases who showed photosensitivity as the sole clinical manifestation, whereas their cellular responses were similar to CS, namely, defective recovery of RNA synthesis and hypersensitivity to killing by UV. Recently, Itoh, Ono, and Yamaizumi15 have described 2 Japanese siblings who were photosensitive without other clinical manifestations of CS, even though their skin fibroblasts showed UV responses characteristic of CS. Furthermore, they assigned these patients and the patient described by Fujiwara et al26 (who is one of the patients reviewed by Cleaver and Thomas25), by complementation analysis, to the same but a new complementation group termed UVs syndrome, which neither belongs to any known groups of CS (groups A and B) nor XP (A through G).15,16 There are no differences between the present case and UVs syndrome in the clinical and cellular characteristics, although they are genetically different. Therefore our patient provides a possibility that UVs syndrome may be a new complementation group of CS, or, at least, a closely related disease. The wide spectrum of clinical manifestations of
Miyauchi-Hashimoto et al 569 CS suggests that biologic and genetic heterogeneity exists. The CS-B gene (also called the ERCC6 gene) has been cloned and characterized.27,28 This revealed that it is a relatively large gene with at least 21 exons and encodes a protein of 168 kd, which contains a DNA helicase motif. Because CS-B cells are defective in the preferential repair of template (transcribed) strands in the actively transcribed genes,7,27,28 the CS-B protein is thought to be involved in the coupling of RNA transcription to excision repair of UV damage. The clinical diversity in CS-B may be due to different mutations occurring at different sites within the CS-B gene, as suggested by the mutational analysis with various group A XP cells.29,30 The present patient without neurologic abnormalities may have a particular mutation in the CS-B gene, distinctly different from mutations detectable in typical CS-B patients. Alternatively, in the present case a mutation could be induced at a certain stage of embryonic development; thus the CS phenotype would be expressed only in restricted organs, such as the skin. We previously reported an evidence of such somatic mosaicism for an XP-A patient.17 In any case, analysis of the mutation in our patient’s cells will solve this problem. REFERENCES 1. Cockayne EA. Dwarfism with retinal atrophy and deafness. Arch Dis Child 1936;11:1-8. 2. Summitt RL. Cockayne syndrome. In: Bergsma D, editor. Birth defect compendium. 2nd ed. New York: Alan R. Liss; 1979. p. 236-7. 3. Lehmann AR. Cockayne’s syndrome and trichothiodystrophy: defective repair without cancer. Cancer Rev 1987;7:82-103. 4. Nance MA, Berry SA. Cockayne syndrome: review of 140 cases. Am J Med Genet 1992;42:68-84. 5. Andrews AD, Barrett SF, Yoder FW, Robbins JH. Cockayne’s syndrome fibroblasts have increased sensitivity to ultraviolet light but normal rates of unscheduled DNA synthesis. J Invest Dermatol 1978;70:237-9. 6. Mayne LV, Lehmann AR. Failure of RNA synthesis to recover after UV-irradiation: an early defect in cells from individuals with Cockayne’s syndrome and xeroderma pigmentosum. Cancer Res 1982;42:1473-8. 7. van Hoffen A, Natarajan AT, Mayne LV, van Zeeland AA, Mullenders LH, Venema J. Deficient repair of the transcribed strand of active genes in Cockayne’s syndrome cells. Nucleic Acids Res 1993;21:5890-5. 8. Barrett SF, Robbins JH, Tarone RE, Kraemer KH. Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells. Mutat Res 1991;255:281-91. 9. Lanning M, Simila S. Cockayne’s syndrome: report of a
Journal of the American Academy of Dermatology October 1998
570 Miyauchi-Hashimoto et al
10. 11.
12. 13.
14. 15.
16.
17.
18. 19.
20.
case with normal intelligence. Z Kinderheilk 1970;109: 70-5. Soffer D, Grotsky HW, Rapin I, Suzuki K. Cockayne syndrome: unusual neuropathological findings and review of the literature. Ann Neurol 1979;6:340-8. Ohta S, Shima A, Shimada M, Katsura T, Nozue T, Ohta S. Ultraviolet sensitivity of Cockayne syndrome and its heterozygotes: cell biological diagnosis of atypical case. Cong Anom 1983;23:399-403. Touge T, Takeuchi H, Miki H, Yamada A, Nishioka M. A case of Cockayne syndrome and analysis of 50 cases in Japan. Intern Med 1986;58:1058-61. (In Japanese) Inoue K, Hanyu N, Takatsu M, Yanagisawa N, Tsukagoshi H. The Cockayne’s syndrome with benign clinical course: a study of four cases from two families. Clin Neurol 1978;18:477-85. (In Japanese) Miyauchi H, Horio T, Akaeda T, Asada A, Chang HR, Ishizaki K, et al. Cockayne syndrome in two adult siblings. J Am Acad Dermatol 1994;30:329-35. Itoh T, Ono T, Yamaizumi M. A new UV-sensitive syndrome not belonging to any complementation groups of xeroderma pigmentosum or Cockayne syndrome: siblings showing biochemical characteristics of Cockayne syndrome without typical clinical manifestations. Mutat Res 1994;314:233-48. Itoh T, Fujiwara Y, Ono T, Yamaizumi M. UVS syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group I. Am J Hum Genet 1995;56:1267-76. Chang HR, Ishizaki K, Sasaki M, et al. Somatic mosaicism for DNA repair capacity in fibroblasts derived from a group A xeroderma pigmentosum patient. J Invest Dermatol 1989;93:460-5. Lehmann AR. Three complementation groups in Cockayne syndrome. Mutat Res 1982;106:347-56. Sugita T, Ikenaga M, Suehara N, Kozuka T, Furuyama J, Yabuuchi H. Prenatal diagnosis of Cockayne syndrome using assay of colony-forming ability in ultraviolet light irradiated cells. Clin Genet 1982;22:137-42. Ayaki H, Hara R, Ikenaga M. Recovery from ultraviolet light-induced depression of ribosomal RNA synthesis in
21. 22. 23.
24.
25. 26.
27.
28.
29.
30.
normal human, xeroderma pigmentosum and Cockayne syndrome cells. J Radiat Res 1996;37:107-16. Noda A, Ikenaga M. Genetic complementation groups of Cockayne syndrome in Japan [abstract]. J Radiat Res 1985;26:52. Horio T. Actinic reticuloid via persistent light reaction from photoallergic contact dermatitis. Arch Dermatol 1982;118:339-42. Brumback RA, Yoder FW, Andrews AD, Peck GL, Robbins JH. Normal pressure hydrocephalus: recognition and relationship to neurological abnormalities in Cockayne’s syndrome. Arch Neurol 1978;35:337-45. Lehmann AR, Thompson AF, Harcourt SA, Stefanini M, Norris PG. Cockayne’s syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation. J Med Genet 1993;30:679-82. Cleaver JE, Thomas GH. Clinical syndromes associated with DNA repair deficiency and enhanced sun sensitivity. Arch Dermatol 1993;129:348-50. Fujiwara Y, Ichihashi M, Kano Y, Goto K, Shimizu K. A new human photosensitive subject with a defect in the recovery of DNA synthesis after ultraviolet-light irradiation. J Invest Dermatol 1981;77:256-63. Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D, Hoeijmakers JHJ. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s syndrome and preferential repair of active genes. Cell 1992;71:939-53. Troelstra C, Hesen W, Bootsma D, Hoeijmakers JHJ. Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne’s syndrome group B. Nucl Acids Res 1993;21:419-26. Satokata I, Tanaka K, Yuba S, Okada Y. Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A xeroderma pigmentosum. Mutat Res 1992;273:203-12. Nishigori C, Moriwaki S, Takebe H, Tanaka T, Imamura S. Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan. Arch Dermatol 1994;130:191-7.
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