Telomere aggregates in trisomy 21 amniocytes

Cancer Genetics and Cytogenetics 195 (2009) 23e26 Telomere aggregates in trisomy 21 amniocytes Efrat Hadia,1, Reuven Sharonyb,c,1, Lilach Goldberg-Bittmanb,d, Tal Biron-Shentala,c, Moshe Fejgina,c, Aliza Amielb,d,* a Department of Obstetrics and Gynecology, Meir Hospital, Kfar Saba, Israel b Genetic Institute, Meir Hospital, Kfar Saba 44281, Israel c Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel d Faculty of Life Science, Bar-Ilan University, Ramat-Gan, Israel Received 12 February 2009; received in revised form 9 March 2009; accepted 10 March 2009 Abstract Trisomy 21 is the most common chromosomal abnormality among persons with intellectual disability, with a live birth rate of 1 in 800e1,000. As such, this abnormality may serve as a model for human disorders that result from supernumerary copies of a genomic region. Down syndrome carries an increased risk of developing acute leukemia and other malignancies. Telomeres of tumor cells nuclei tend to form aggregates (TA). This study evaluated TA formation in amniocytes from trisomy 21 pregnancies, compared with amniocytes from normal euploid pregnancies. A commercially available peptide nucleic acid telomere kit was used to evaluate TA formation, using two-dimensional fluorescence microscopy. Significantly higher frequencies of TA were found in trisomy 21 amniocytes than in amniocytes from normal pregnancies. The TAs found in trisomy 21 amniocytes apparently represent an additional parameter that reflects the high genetic instability of this syndrome and its recognized predisposition to develop leukemia and other malignancies. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Chromosomal abnormalities occur with astonishing frequency in humans, and are present in 50e60% of embryos and early fetuses that are spontaneously aborted [1]. Of the different classes of chromosomal abnormalities, aneuploidy (primarily as trisomy and monosomy) is by far the most common and, clinically, the most important. The majority of aneuploid fetuses are nonviable [2]; those surviving to term account for 0.9% of live births. Although only about one fourth of trisomy 21 conceptuses survive to birth [3], this is still the most common genetic cause for congenital malformations and intellectual impairment, occurring in 1 of every 800e1,000 live births [4]. As such, this abnormality may serve as a model for human disorders that result from supernumerary chromosome copies of a genomic region. Trisomy 21 gives rise to a variety of traits, all of which have variable penetrance and clinical expressivity [5,6]. Individuals with Down syndrome (DS) are at increased risk of developing leukemia * Corresponding author. Tel.: þ972-9-747-2220; fax: þ972-9-7471296. E-mail address: amielaliza@clalit.org.il (A. Amiel). 1 Both authors contributed equally to the research. 0165-4608/09/$ e see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.03.003 [7] and other malignancies [8]. This has been attributed to the primary aneuploidy (trisomy 21) in these individuals, although the actual mechanism is not clear. Telomeres are TTAGGG repetitions at the chromosomes ends. Functional telomeres are essential for the normal segregation and maintenance of chromosomes during mitotic and meiotic divisions [9]. Maintenance of telomeres depends on interaction with the enzyme telomerase, with telomeric proteins, and with some still-undiscovered factors that regulate telomere functions. Dysfunctional telomeres support the survival of aneuploid cells, a characteristic of many human malignancies [10]. A remarkable difference between normal and tumor cells becomes apparent when a three-dimensional (3-D) imaging approach is applied: a specific 3-D telomeric signature is revealed for tumor cells. In contrast to the nonoverlapping nature of telomeres in normal nuclei, telomeres of tumor nuclei tend to form aggregates (TAs). Various numbers and sizes of such TA can be found in tumor nuclei [11]. The formation of TA is independent of telomere length and telomerase activity [12]. At least two types of telomeric dysfunction are described in tumor cells. One type involves the formation of TA; the other involves critically short telomeres [10]. Both types of telomeric dysfunction can lead to a breakageebridgeefusion 24 E. Hadi et al. / Cancer Genetics and Cytogenetics 195 (2009) 23e26 cycle that contributes to deletions, gene amplification, nonreciprocal translocation, and other genetic changes associated with tumor genesis [13e15]. Analysis of primary tumors indicates that TAs are common. Various cell types and tissues have been examined, including primary head and neck cancer, primary mouse plasmacytoma, human neuroblastoma, and colon carcinoma cell lines [11]. Earlier studies suggest that such changes in the telomeric organization of the interphase nucleus may occur before the cells become neoplastic. For example, in cervical cancer, noninvasive lesions (e.g., cervical intraepithelial neoplasia I) show TA in some of the cells. Our research group has reported higher TA rates in different stages of non-Hodgkin lymphoma [16] and in chronic hepatitis C patients [17]. Given this apparent predisposition to developing cancer, our objective was to evaluate TA frequencies in amniocytes from trisomy 21 pregnancies, compared with those from normal diploid pregnancies. observation. The slides were scored blindly by two readers, and 100 cells in interphase were analyzed per case. 2.3. Telomere aggregate count Aggregate size was divided into three groups, relative to the size of a single telomere: (1) fusion of 2e5 telomeres; (2) fusion of 6e10 telomeres; and (3) fusion of 11e15 telomeres. For the purpose of statistical analysis, the different groups were scored as follows: group 1, one point; group 2, two points; and group 3, three points. The measurements were done using CytoVision software (Applied Imaging, Santa Clara, CA). The slides were scored blindly. 2.4. Statistical analysis A two-tailed sample t-test was applied for testing differences between the study groups, with P < 0.05 considered statistically significant. Microsoft Excel software (office 2000) was used for statistical analysis. 2. Materials and methods 2.1. Amniocyte cell cultures Twenty human amniotic-fluid cell cultures were established after amniocentesis diagnosis at our institute: 10 cultures from normal diploid amniocytes and 10 cultures from trisomy-21 amniocytes. All cases had been diagnosed during the same time period. The cells were grown in 25-mL tissue-culture flasks at 37 C in 5% CO2 with F10 medium (Biological Industries, Beit Haemek, Israel), supplemented with 20% fetal calf serum (Biological Industries), 1% glutamine, and 1% antibiotics, until ~75% confluent and still actively dividing. Cells were harvested with trypsin (SigmaeAldrich, St. Louis, MO), treated with 0.075 mol/L KCl at 37 C for 15 minutes and washed three times with fresh, cold 3:1 methanoleacetic acid. The amniocyte suspensions were stored at 20 C until use. All cell samples used were taken from the original culture flasks without any passage. 2.2. Telomere fluorescent in situ hybridization Telomere lengths in cells were assessed by the fluorescent intensity of telomere fluorescence in situ hybridization for each slide, using a CY3-labeled telomere-specific peptide nucleic acid probe (catalog no. 5326; DAKO, Glostrup, Denmark). The slides were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (1,000 ng/mL, Vysis catalog no. 32-804830; Abbott Molecular, Des Plaines, IL), and finally overlaid with glass cover slips for 3. Results When nuclei are analyzed in 3-D microscopy, no overlapping TA are expected in normal material, whereas overlapping is expected to some extent as an artifact in 2-D analyses of normal material. Because we were using a 2-D system, we first established the baseline rate of TA in the control group, which was found to be 1.68 aggregates per nucleus. These background TAs are considered to be a technical artifact, due to a virtual overlap of telomeres within the 2-D nucleus that cannot be distinguished under a 2-D microscope. We found significantly more TA in the trisomy 21 cells (mean number, 10.36; P ! 0.01) than in the control group (Fig. 1). The number of all three categories of TAs per cell was higher in the amniocytes of trisomy 21 cases than in control cases. 14 12 mean number of telomere aggregats This study was conducted under an institutional review boardeapproved protocol. The study group consisted of 10 cases of trisomy 21 and the control group consisted of 10 cases with normal diploid karyotype. Control Trisomy 21 10 8 6 4 2 0 Fig. 1. The mean number of telomeres aggregates (TA) in trisomy 21 patients and the control group. Error bars indicate standard error. E. Hadi et al. / Cancer Genetics and Cytogenetics 195 (2009) 23e26 4. Discussion Greater numbers of TA were found in amniocytes taken from patients with trisomy 21 fetuses, compared with amniocytes from normal diploid fetuses. Telomere aggregates have been observed in both malignant and premalignant conditions [18]. We have found it also in a chronic hepatitis C patient, which is considered to be a premalignant disease [17]. At the present stage of research, there appears to be a correlation between TA formation and malignant potential, but the prognostic value is yet to be established. Down syndrome patients exhibit a higher rate of telomere loss, compared with age-matched controls [19]. Recently, Jenkins et al. [20,21] reported that telomere length in T lymphocytes of individuals with DS and dementia or mild cognitive impairment (MCI-DS) is reduced, compared with that in individuals with DS but without dementia or MCI-DS. The authors suggested that counting chromosome ends for the presence or absence of fluorescent signal might provide a valid biomarker of dementia status [21]. Down syndrome carries a 10- to 30-fold increased risk of developing acute leukemia [7]. Peak incidence is in the newborn period (10%) and in early childhood (3e6 years of age) [22], but increased risk of developing leukemia extends into adulthood [23]. Other malignancies that occur more frequently in DS patients are germ cell tumor and, perhaps, gastrointestinal cancers. Solid tumors, on the other hand, are less frequent in this group of patients [23,24]. Cytogenetic studies of leukemia cells in DS patients suggest genetic pathways at different levels to be involved in the leukemogenic process, including GATA1 mutations and various chromosomal aberrations and supernumerary chromosomes [25]. Shorter telomere length could represent one of the cytogenetic parameters correlated with both the risk of developing dementia and myeloproliferative disorders in individuals with DS. Telomere aggregate formation could represent another one. The high variation of the aggregates could also be a reflection of the set of aneuploidic genes, which exhibits greater variance of expression in human trisomy 21 tissues than in euploid tissues, as well as their effect on the euploid gene dosage [26]. These characteristics may contribute to producing the variable phenotypic abnormalities observed in DS. We have previously reported that loss of replication control is one of the characteristics of the aneuploidic syndromes with trisomy 21, 18, and 13 [27]. Such loss of replication control could be due to the effect of abnormal dosage of normal genes generated by the increased number of chromosomes. Two other aspects of genetic instability reported in cells from patients with DS [28] are random aneuploidy and the telomere capture phenomenon. Both have been shown to be present also in malignant cells [29]. 25 It thus seems that these multifactorial disorders arise because of interference with the mechanism and control of gene replication. This supports the hypothesis, presented in the review by Birchler et al. [26], that ‘‘aneuploidic syndromes are likely to reflect an upset in the balance of the regulators that modulate target genes throughout the genome, which in turn will change the phenotype.’’ Recent findings, however, suggest that this balance involves the regulatory system more than changes in the dosage of the target genes, as is often envisaged. A reduced level of expression from crucial target genes, as a result of regulatory imbalance, might have phenotypic effects in both monosomies and trisomies [30,31]. It seems that the presence of the TA found in trisomy 21 amniocytes represents an additional parameter reflecting the high genetic instability of this syndrome and its recognized predisposition to develop leukemia and other malignancies. References [1] Kajii T, Ferrier A, Niikawa N, Takahara H, Ohama K, Avirachan S. Anatomic and chromosomal anomalies in 639 spontaneous abortuses. Hum Genet 1980;55:87e98. [2] Snijders RJ, Sebire NJ, Nicolaides KH. 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