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Pediatric Hematology and Oncology, 20: 651–656, 2003 C Taylor & Francis Inc. Copyright ISSN: 0888-0018 print / 1521-0669 online DOI: 10.1080/08880010390243086
Short Report
SENSITIVITY TO RADIATION AND ALKYLATING AGENT OF PERIPHERAL LYMPHOCYTES AND FIBROBLASTS IN A HOYERAAL–HREIDARSSON SYNDROME PATIENT
Radhia M’kacher ✷ Laboratoire de Radiosensibilit´e-radiocarcinogen`ese, Institut Fustave Roussy, Villejuif, France; and D´epartement de Medecine, Institut Fustave Roussy, Villejuif, France Veronique Laithier ✷ Service de P´ediatrie, Centre Hospitalier Universitaire de Besancon, Bescancon, France Alexandre Valent ✷ Laboratoire de Cytog´en´etique et G´en´etique Oncologique, Institut Fustave Roussy, Villejuif, France Francois Delhommeau
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INSERM, Institut Fustave Roussy, Villejuif, France
Dominique Violot, Eric Deutsch, Julien Dossou, Nadine B´eron–Gaillard, and Theodore Girinsky ✷ Laboratoire de Radiosensibilit´e-radiocarcinogen`ese, Institut Fustave Roussy, Villejuif, France Jean Bourhis ✷ Laboratoire de Radiosensibilit´e-radiocarcinogen`ese, Institut Fustave Roussy, Villejuif, France; and D´epartement de Medecine, Institut Fustave Roussy, Villejuif, France Patrice Carde ✷ D´epartement de M´edecine, Institut Fustave Roussy, Villejuif, France Alain Bernheim ✷ Laboratoire de Cytog´en´etique et G´en´etique Oncologique, Institut Fustave Roussy, Villejuif, France Claude Parmentier ✷ Laboratoire de Radiosensibilit´e-radiocarcinogen`ese, Institut Fustave Roussy, Villejuif, France
Received 26 March 2003; accepted 8 July 2003. We are grateful to Tom J. Vulliamy (Imperial College School of Medicine, London) for the screening of DKC1 gene and for his suggestions. We thank Danielle Fenneux (Hopital Kremlin Bicˆetre) for the telomere length investigation and Nicole Chavaudra and Bernard Clausse for their participation in this study. We also thank Ingrid Kuchenthal for preparing the manuscript. This work was supported by grants from the Gustave Roussy Institute (CRC 98-25 and CRC 2000-01) and Electricit´e de France (EDF 98-04). Address correspondence to Dr. Radhia M’kacher, Institut Gustave Roussy, 94 805 Villejuif, France. E-mail:
[email protected]
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R. M’kacher et al. ✷ Hoyeraal–Hreidarsson syndrome (HHS) is a severe infantile variant of X-linked dyskeratosis congenita (DC). The authors report evidence of increased in vitro sensitivity to radiation and alkylating agent in circulating lymphocytes and fibroblasts obtained from a 7-year-old boy with HHS. A major telomere shortening was also found (3 kb) as compared to healthy donors (10 kb). The standard treatments, chemotherapy regimens, and radiation therapy were not possible. The data suggest that conditioning regimens including TBI should not be used when a bone marrow transplantation procedure is planned in these patients.
Keywords. bone marrow transplantation, Hoyeraal–Hreidarsson syndrome, radiation sensitivity, telomere length
Hoyeraal–Hreidarsson syndrome (HHS), a severe multisystem disorder affecting boys, is characterized by microcephaly, cerebellar hypoplasia, growth retardation of prenatal onset, and aplastic anemia [1, 2]. The pathogenesis of HHS is unknown. A recent study [3] has shown that the DKC1 gene, located on Xq28 and encoding the dyskerin nuclear protein, was mutated in HHS patients. This mutation demonstrated that HHS is a severe variant of X-linked dyskeratosis congenita (DC) [4]. In this study, we report on the treatment of a boy with HHS. We assessed the sensitivity to ionizing radiation and alkylating agent of circulating lymphocytes and primary fibroblasts to detect DNA repair abnormalities leading to increased constitutive sensitivity. The status of the DKC1 gene and telomere length were studied in relation to these findings. Previous study has demonstrated an enhanced G2 chromatid radiosensitivity in cultured DC fibroblasts [5]. Similarly, telomere shortening has been observed in peripheral blood of DC patients [6]. The standard treatment was modified based on previous reports, in vitro sensitivity to alkylating agent, and ionizing radiation of cultured lymphocytes and fibroblasts.
CASE REPORT We studied the sensitivity to ionizing radiation and alkylating agent in a 7-year-old HHS boy presenting with intrauterine growth retardation (birth weight: 2.4 kg) growth failure (−3 SD), microcephaly, and developmental delay. The head MRI showed an atrophy of the cerebellum. At 42 months, he presented with a thrombopenia (34 × 109 /L) and 9 months later, bone marrow aspiration showed an hypocellular status. Increase of pancytopenia led to the decision of bone marrow transplantation with his HLA identical sister, after chemotherapy conditioning (endoxan: 10 mg/kg/J; fludarabine 40 mg/m2 /J).
METHODS Peripheral blood samples, fibroblasts were irradiated in vitro with 0.50 Gy/min 220 kV X-rays at different doses (2 and 4 Gy). The same procedure was applied to control populations composed of healthy donors (HD)
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(n = 20; mean age: 40 years (19–66 years)) and radiation-sensitive lymphoma patients (LP) who have developed a huge reaction after radiation treatment (n = 2; mean age: 25 years (17–33 years)). Lymphocyte culture was performed and mechlorethamine (0.05 µg/mL) sensitivity was measured following the Fanconi anemia sensitivity assay protocol [7]. R-banding karyotyping was established. Clonogenic assay was performed to assess the radiation sensitivity of fibroblasts [8], and FISH technique was used to score cytogenetic abnormalities [9]. To estimate telomere length, slides from patient (before and after treatment) were hybridized with PNA probe (Kit PNA FISH/Cy3 DAKO, Glostrup, Denmark). Fluorescent signal was quantified on metaphase and interphase nuclei with an image analysis system [10]. Results were expressed as the mean intensity of fluorescence per spot.
RESULTS Cytogenetic and Molecular Investigations No karyotype aberration was found by R-banding or multicolor FISH (M-FISH) karyotyping. SSCP screening of the DKC1 gene showed fragments of normal mobility except for exon 14 presenting a shift characteristic of the cDNA residue 1461 C → T polymorphism [3]. Using Q-FISH technique, we showed that the HHS patient presented (before and after treatment) a very weak mean fluorescence intensity/spot on both metaphases on (178.8–103.8, n = 12) and nuclei (140.3–162.2, n = 25) compared to healthy donors. An important decrease in the mean intensity of fluorescence/spot was 79% for metaphases from healthy donors and 78% for their nuclei. Note that a TRF length of 3.4 kb (Southern blot) of our controls is related to a mean intensity of fluorescence (222.0–107.6), suggesting a very short telomere length in the lymphocytes of the HHS patient (around 3 kb).
Cell Survival Figure 1 shows the severe radiation sensitivity phenotype of the fibroblasts of HHS patient compared to fibroblasts from healthy donors and lymphoma patients. The surviving fraction at 2 Gy (SF2) was 30% for HD, 18% for LP, and 6% for the HHS patient. SF2 was 5-fold reduced in HHS patients as compared to healthy donors. The lymphocytes were also processed for the measurement of their capacity to repair radiation-induced DNA double-strand breaks. After 2-Gy in vitro irradiation, the number of mitoses was depressed. The frequency of chromosomal aberrations in circulating lymphocytes was 3-fold higher in the HHS patient (1.07 [95% confidence interval (CI): 0.84–1.35] aberrations per cell) than in HD (0.28 [95% CI: 0.20–0.30] per cell) (Figure 2).
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FIGURE 1 Clonogenic survival assay demonstrates the presence of a significant radiation sensitivity of an HHS patient as compared to healthy donors and lymphoma patients.
FIGURE 2 Frequency of chromosomal abnormalities in circulating lymphocytes before and after in vitro irradiation at 2 Gy in an HHS patient, radiation-sensitive lymphoma patients, and healthy donors. Chromosome 1, 3, and 4 painting was applied to score the induced chromosomal aberrations.
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The lymphocytes of the HHS patient showed a higher sensitivity to alkylating agent (mechlorethamine) than those of Fanconi anemia patient. At the same concentration of mechlorethamine, lymphocyte culture did not yield any metaphase and the lymphocytes appeared to be dead. On the other hand, metaphases were obtained in Fanconi anemia lymphocyte culture, and acentric fragments were scored.
DISCUSSION We demonstrated in this study the increased sensitivity to ionizing radiation and alkylating agent in an HHS patient. A higher radiation sensitivity in HHS patient fibroblasts was compared to that observed in ataxia telangectasia fibroblasts (SF2 = 2.5%) [8]. Circulating lymphocytes responded similarly, exhibiting significantly elevated frequencies of chromosomal aberrations compared to normal human lymphocytes (3-fold). We confirmed the 3-fold increase in the in vitro radiation sensitivity of our patient at the level of both cellular sensitivity (Figure 1) and chromosomal aberration processing (Figure 2). In addition, a higher chemo-sensibility to alkylating agent has been demonstrated. This radiochemosensitivity was correlated to telomere shortening, confirming the role of telomere length in radiation sensitivity and identifying telomere shortening as one of the characterized genetic disorders. The very short telomere length associated with the high incidence of chromosomal abnormalities after in vitro irradiation observed in this young HHS patient suggests a possible link between telomere size maintenance and DNA doublestrand break (DSB) repair. A similar significant correlation between telomere length and chromosomal radiation sensitivity was also observed in lymphocytes from Hodgkin lymphoma patients [11]. In the HHS patient, standard treatment regimens were not available, including TBI and chemotherapy. This hypersensitivity to alkylating agent was subsequently used to reduce the dose of the patient’s chemotherapy course before bone marrow transplantation. Also, the HHS patient received only chemotherapy comparable to that used in the Fanconi anemia patient. The radiation therapy was lightened at the level of cellular hyperradiation sensitivity comparable to that observed in AT patients. This observation is of a great interest because (1) it shows a clear link between telomere shortening, induced chromosomal aberrations, and radiation sensitivity [12–14]; (2) it demonstrates the possibility of establishing a prognostic cellular index of radiochemosensitivity in HHS patients. This radiochemosensitivity could be important in developing clinical therapeutic strategy.
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REFERENCES 1. Hoyeraal HM, Lamvik J, Moe PJ. Congenital hypoplastic thrombocytopenia and cerebral malformations in two brothers. Acta Paediatr Scand. 1970;59:185–191. 2. Hreidarsson S, Kristjansson K, Johannesson G, Johannsson JH. A syndrome of progressive pancytopenia with microcephaly, cerebellar hypoplasia and growth failure. Acta Paediatr Scand. 1988;77:773– 775. 3. Knight SW, Heiss NS, Vulliamy TJ, et al. Unexplained aplastic anaemia, immunodeficiency, and cerebellar hypoplasia (Hoyeraal–Hreidarsson syndrome) due to mutations in the dyskeratosis congenita gene, DKC1. Br J Haematol. 1999;107:335–339. 4. Dokal I. Dyskeratosis congenita in all its forms. Br J Haematol. 2000;110:768–779. 5. DeBauche DM, Pai GS, Stanley WS. Enhanced G2 chromatid radiosensitivity in dyskeratosis congenita fibroblasts. Am J Hum Genet. 1990;46:350–357. 6. Vulliamy TJ, Knight SW, Mason PJ, Dokal I. Very short telomeres in the peripheral blood of patients with X-linked and autosomal dyskeratosis congenita. Blood Cells Mol Dis. 2001;27:353–357. 7. Berger R, Bernheim A, Le Coniat M, Vecchione D, Schaison G. Nitrogene mustard-induced chromosome breakage: a tool for Fanconi’s anemia diagnosis. Cancer Genet Cytogenet. 1980;2:269–274. 8. Badie C, Alsbeih G, Reydellet I, Arlett C, Fertil B, Malaise EP. Dose-rate effects on the survival of irradiated hypersensitive and normal human fibroblasts. Int J Radiat Biol. 1996;70:563–570. 9. M’Kacher R, Schlumberger M, Legal JD, et al. Biologic dosimetry in thyroid cancer patients after repeated treatments with iodine-131. J Nucl Med. 1998;39:825–829. 10. Martens UM, Brass V, Engelhardt M, et al. Measurement of telomere length in haematopoietic cells using in situ hybridization techniques. Biochem Soc Trans. 2000;28:245–250. 11. M’kacher R, Bennaceur-Griscelli A, Girinsky T, et al. Detection of DNA repair abnormalities and telomere shortening in circulating lymphocytes: relevance to predict late complications and secondary malignancies in Hodgkin’s lymphoma patients. 2002; Blood. 100:90a. 12. Bertinato J, Schild-Poulter C, Hache RJ. Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits. J Cell Sci. 2001;14:89–99. 13. Goytisolo FA, Samper E, Martin-Caballero J, et al. Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med. 2000;192:1625–1636. 14. Kilburn AE, Shea MJ, Sargent RG, Wilson JH. Insertion of a telomere repeat sequence into a mammalian gene causes chromosome instability. Mol Cell Biol. 2001;21:126–135.
