Chromosoma (2005) 114: 118–126 DOI 10.1007/s00412-005-0343-7
RESEARCH ARTICLE
David Gisselsson . Chunbo Shao . Cathy M. Tuck-Muller . Suzana Sogorovic . Eva Pålsson . Dominique Smeets . Melanie Ehrlich
Interphase chromosomal abnormalities and mitotic missegregation of hypomethylated sequences in ICF syndrome cells Received: 21 December 2004 / Revised: 29 March 2005 / Accepted: 29 March 2005 / Published online: 27 April 2005 # Springer-Verlag 2005
Abstract The immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease. Usually, it is caused by mutations in the DNA methyltransferase 3B gene, which result in decreased methylation of satellite DNA in the juxtacentromeric heterochromatin at 1qh, 16qh, and 9qh. Satellite II-rich 1qh and 16qh display high frequencies of abnormalities in mitogen-stimulated ICF lymphocytes without these cells being prone to aneuploidy. Here we show that in lymphoblastoid cell lines from four ICF patients, there was increased colocalization of the hypomethylated 1qh and 16qh sequences in interphase, abnormal looping of pericentromeric DNA sequences at metaphase, formation of bridges at anaphase, chromosome 1 and 16 fragmentation at the telophase–interphase transition, and, in apoptotic cells, micronuclei with overrepresentation of chromosome 1 and 16 material. Another source of anaphase bridging in the ICF cells was random telomeric associations between chromosomes. Our results elucidate the mechanism of formation of ICF chromosome anomalies and suggest that 1qh–16qh associations in interphase can lead to disturbances of mitotic segregation, resulting in micronucleus formation and Communicated by S. Gerbi D. Gisselsson . E. Pålsson Department of Clinical Genetics, University Hospital, 221 85 Lund, Sweden C. Shao . M. Ehrlich (*) Human Genetics Program and Department of Biochemistry, Tulane Medical School, New Orleans, LA, 70112, USA e-mail:
[email protected] Tel.: +1-504-9882449 Fax: +1-504-9881763 C. M. Tuck-Muller . S. Sogorovic Department of Medical Genetics, University of South Alabama, Mobile, AL, 36688, USA D. Smeets Department of Human Genetics, University Medical Center St. Radboud, Nijmegen, The Netherlands
sometimes apoptosis. This can help explain why specific types of 1qh and 16qh rearrangements are not present at high frequencies in ICF lymphoid cells despite diverse 1qh and 16qh aberrations continuously being generated. Abbreviations DAPI: diamidinophenylindole . DNMT3B: DNA methyltransferase 3B . EBV: Epstein–Barr virus . FISH: fluorescence in situ hybridization . ICF: immunodeficiency, centromeric instability, facial anomalies . LCL: lymphoblastoid cell line . Sat2: classical satellite II DNA . MN: micronuclei . PBS: phosphate-buffered saline
Introduction The immundeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease. Since it was first described by Hulten et al. (1978), about 35 cases have been reported in the literature. It has recently been shown that most ICF patients exhibit inactivating mutations of the DNA methyltransferase 3B gene (DNMT3B), leading to invariant hypomethylation of a limited number of genomic regions (Kondo et al. 2000), in particular the heterochromatic regions containing tandemly repeated sequences of classical satellite II (Sat2) and III (Sat3) (Jeanpierre et al. 1993; Hansen et al. 1999; Xu et al. 1999). Immunologically, ICF patients show hypogammaglobulinaemia but with B-cells present (Ehrlich 2003). It has been suggested that this condition is mediated by the dysregulation of lymphogenesis genes (Ehrlich et al. 2001) and defective B-cell differentiation, leading to the accumulation of immature B-cells with an increased rate of apoptosis upon in vitro activation (Blanco-Betancourt et al. 2004). Because of a strong predisposition for systemic infectious diseases, the majority of ICF patients die before reaching their teens. Peripheral blood lymphocytes from ICF patients are cytogenetically characterized by abnormalities of the long Sat2-rich pericentromeric heterochromatin regions of chromosome 1 and 16, and, to a much lesser extent, also of the
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Sat3-rich or Sat2-rich heterochromatic regions of chromosomes 9, 2, and 10 (Ehrlich 2003). The abnormalities of chromosomes 1 and 16 include decondensation (stretching) of the heterochromatin, whole-arm deletions and translocations, isochromosome formations, and multiradial chromosomes. Consistent with DNA demethylation causing these abnormalities, the DNA methylation inhibitor 5-azacytidine has been shown to induce ICF-like decondensation and rearrangements targeted to the 1qh, 16qh, and 9qh regions in normal lymphoid cells (Schmid et al. 1983; KokaljVokac et al. 1993; Hernandez et al. 1997). The cytogenetic changes are typically present in mitogenically stimulated Band T-cells from peripheral blood of ICF patients, whereas they are less frequent or less complex in bone marrow cells and fibroblasts (Tiepolo et al. 1979; Carpenter et al. 1988; Maraschio et al. 1988; Turleau et al. 1989; Fasth et al. 1990; Smeets et al. 1994; Brown et al. 1995). The reason for this tissue-specificity is not known. It has been suggested that DNMT3B plays a role in 1qh and 16qh condensation by interacting with other proteins in the condensin complex and with DNA motor proteins (Geiman et al. 2004). However, early embryogenesis proceeds normally in DNMT3B knockout mice and DNMT3B is usually much less plentiful in postnatal than embryonic cells (Okano et al. 1999). There is still a lack of experimental data regarding several cytogenetic aspects of the ICF syndrome. It is not known when in the cell cycle the different chromosome aberrations are generated. Neither is it known whether the cytogenetic abnormalities are created independently of each other or if some of the abnormalities arise as derivatives of other abnormalities. This study is an attempt to answer these questions by monitoring the interphase configuration of hypomethylated DNA sequences in lymphoblastoid cell lines (LCLs) from four ICF syndrome patients, as well as their chromosome dynamics at mitosis and the frequency of genomic imbalances among apoptotic cells. Our findings suggest that illegitimate recombination of heterochromatic sequences at interphase due to increased pericentromeric associations of chromosomes 1 and 16 leads to severe perturbations of the mitotic process, resulting in a variety of abnormal chromosome derivatives, and an increased propensity for apoptosis among genetically unbalanced cells. Surprisingly, the mitotic abnormalities were enhanced in three of the four ICF LCLs by the presence of short dysfunctional telomeres among various chromosomes.
Materials and methods Cell culture, chromosome dynamics, and chromosome banding Four LCLs (ICF K, ICF C, ICF B, and ICF P5) obtained from Epstein–Barr virus (EBV)-transformed peripheral blood B-cells of ICF patients were used (Carpenter et al. 1988; Kieback et al. 1992; Smeets et al. 1994; Wijmenga et al. 2000). Two EBV-transformed LCLs from healthy individuals (AG15022 and AG14953; Coriell Institute) were the normal controls. The cells were grown in RPMI 1640
medium with HEPES buffer, supplemented with 15% (ICF K) or 20% (ICF B, ICF C, ICF P5, AG15022, AG14953) fetal bovine serum, 0.23 mg/ml L-glutamine, 100 IU/ml penicillin, and 0.2 mg/ml streptomycin. For analysis of metaphase chromosomes, cells were arrested at metaphase with 0.02 μg/ml Colcemid, harvested, and G-banded by standard procedures. The structure of heterochromatin was assessed in a blinded fashion in 50 metaphase cells from each LCL. Decondensation was scored when the entire length of 1qh or 16qh was thread-like with the width of the heterochromatin of both chromatids being no greater than that of the centromere of the same chromosome (TuckMuller et al. 2000). Visualization of chromosome dynamics at mitosis was performed by harvesting without metaphase arrest, washing in phosphate-buffered saline (PBS), fixation in 3:1 methanol:acetic acid, followed by hematoxylin– eosin staining. Anaphase cells showing at least one string of chromatin connecting the poles were classified as harboring an anaphase bridge. Lagging chromosomes were defined as bodies staining with hematoxylin or diamidinophenylindole (DAPI) in a fashion similar to the mitotic chromosomes in the same cell, but being clearly separated from the metaphase or anaphase plates by at least one chromosome width (approximately 1 μm). Micronuclei (MN) were defined as hematoxylin or DAPI-positive bodies adjacent to a nucleus and with the same staining as that nucleus, but with a diameter no larger than one third of the maximum nuclear diameter. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) was performed as previously described (Gisselsson et al. 2000). Cell-division preparations harvested without metaphase arrest and hypotonic treatment were treated with 20 mg/ml pepsin for 10 min in 0.01 M HCl and fixed in 1% formalin/PBS prior to hybridization. Centromeric satellite sequences were detected using a Cy3-labelled human pan-alpha satellite probe (CAMBIO, Cambridge, UK). Commercially available probes (Vysis/Abbott Diagnostics, Abbott Park, IL) were used for whole-chromosome painting and detection of Sat2 sequences of chromosomes 1 or 16. Telomeric TTAGGG repeats were visualized by FISH with fluorescein-conjugated (CCCTAA)3 peptide nucleic acid probes (Landsdorp et al. 1996). The signal intensity was directly quantified by the Cytovision software (Applied Imaging, Newcastle, UK), and the number of negative chromosome termini for each metaphase cell was scored. Although FISH-negative chromosome ends may still contain up to 500 bp of telomeric repeats, this method gives a valid assessment of the protective capacity of individual telomeres (Martens et al. 2000; Gisselsson et al. 2001). Chromosomes were counterstained with DAPI. For the examination of interphase cells for colocalization of 1qh and 16qh signals with duallabeled probes, we used only cells with nuclei that had two 1qh signals and two 16qh signals, including those with an overlapping 1qh and 16qh signal; therefore, only cells in G1 or early or middle S-phase were examined.
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In each of the ICF LCLs, 18–40% of the metaphase cells displayed at least one chromosome 1 or 16 homologue with decondensation in the qh region in a variety of configurations (Fig. 1a). For example, in ICF C there were five metaphases with a single 1qh region decondensed, two with both allelic 1qh regions decondensed, one with both 1qh regions and a single 16qh region decondensed, and one with a single 1qh and both 16qh regions decondensed. Moreover,
4–16% of the cells had rearrangements in 1qh and/or 16qh. These pericentromeric rearrangements of chromosome 1 or 16 consisted of whole-arm deletions, chromosome breaks (Fig. 1a), multiradials, and translocations (Table 1). Most of these were nonclonal, i.e., each specific type of rearrangement was observed in less than two metaphase cells. For example, in 50 metaphases from ICF C, there was one cell each with a quadriradial(1:16)(pq;pq), a quadriradial (16;16)(pq;pq), and with del(1)(q12). The chromosome 1 and 16 abnormalities were not seen in the control LCLs although they have been observed at low frequencies in other control LCLs (Tuck-Muller et al. 2000). Although non-clonal gains and losses of whole chromosomes were found in some of the ICF LCLs, there was no overrepresentation of chromosomes 1 and 16 among these imbalances and they did not occur at a high frequency. As shown in Table 1, telomere associations were found much more frequently in the ICF B, ICF C, and the ICF P5 LCLs (one to two per positive metaphase cell; Fig. 1b) than in ICF K and the control LCLs. Compared to AG15022, which had the higher frequency of telomeric fusions of the two control LCLs (6%), the ICF B, ICF C, and ICF P5
Fig. 1 a Decondensation of the 1qh heterochromatin in one homologue (arrow) and fission of the arms of the other chromosome 1 homologue (arrowheads pointing to separated 1p and to 1q) in ICF C. b Telomeric association (arrow) in ICF C. c, d Anaphase bridges in
ICF P5. e Interphase chromatin bridge in ICF C. f Lagging chromosome material at metaphase in ICF C. g Looping chromatin strings at metaphase in ICF B. h, i Apoptotic nuclei in ICF B with chromatin strings, probably representing vestiges of anaphase bridges
Detection of apoptotic cells Phosphatidylserine on the outer leaflet of the cytoplasmic membrane was detected by Annexin V staining (Vybrant Apoptosis Kit, Molecular Probes, Eugene, OR). Fragmented DNA was fluorescently labeled using terminal transferase (TUNEL assay; In Situ Cell Death Detection Kit, Roche, Mannheim, Germany).
Results Chromosome aberrations at metaphase
121 Table 1 Chromosome banding data and telomere status Cell line
ICF K ICF C
Karyotypesa
46,X,der(Y)t(Y;3)(q12;q12) 46,XX,r(19)(p13;q13)[2]/ 46,XX,tas(13;22)(p13;p13)[2]/ 46,XX,tas,(19;22)(p13;q13)[2]/ 46,XX[94] ICF B 46,XY,tas(13;15)(p13;p13)[3]/ 46,XY[97] ICF P5 46,XY AG15022 46,XY AG14953 46,XY
Percentage of metaphases (actual ratio)b
TTAGGGnegative 1qh or 16qh 1qh or 16qh Non-clonal Non-clonal Telomere decondensation rearrangement chromosome chromosome associations termini: mean valuec losses gains 40 (20/50) 24 (12/50)
4 (2/50) 6 (3/50)
8 (4/50) 2 (1/50)
4 (2/50) 2 (1/50)
2 (1/50) 22 (11/50)
1.9 3.7
18 (9/50)
4 (2/50)
2 (1/50)
0 (0/50)
44 (22/50)
4.5
36 (18/50) 0 (0/50) 0 (0/50)
16 (8/50) 0 (0/50) 0 (0/50)
0 (0/50) 0 (0/50) 0 (0/50)
0 (0/50) 0 (0/50) 0 (0/50)
34 (17/50) 6 (3/50) 2 (1/50)
4.7 2.1 1.8
a
One hundred metaphases were scored for the karyotypes. Although the karyotypes of ICF B, ICF C, and ICF K at an earlier passage were different, as described in a previous report (Ehrlich et al. 2001), e.g. ICF C had a 46 XX phenotype, the metaphase abnormalities at 1qh and 16qh were similar in the previous study and the present one. Furthermore, previously we demonstrated similar 1qh and 16qh anomalies in ICF B metaphases in tetraploid and diploid metaphases b Percentage of metaphases with at least one abnormality; 50 metaphases were scored for 1qh, 16qh, and non-clonal abnormalities as well as telomere associations c Mean value of at least ten cells
LCLs had a statistically significant overrepresentation of metaphase cells containing telomeric fusions (0.0001
