LEAD ARTICLE The Cytogenetics of Ataxia Telangiectasia Tracy L. Kojis, Richard A. Gatti, and Robert S. Sparkes

ABSTRACT: Ataxia-telangiectasia (AT) is a heterogeneous autosomal recessive disorder marked by cerebellar ataxia, oculocutaneous telangiectases, hypersensitivity to ionizing radiation, immunodeficiency, and cancer susceptibility. AT is also a spontaneous chromosomal breakage syndrome, notable for tissue-specific cytogenetic changes and telomeric fusions. Molecular characterization of rearrangements specific to T-lymphocytes suggests that a DNA repair~processing defect is potentially responsible for the diverse array of chromosomal abnormalities observed in a variety of AT cell types.

INTRODUCTION

Ataxia-telangiectasia (AT), a rare autosomal recessive disorder, belongs to a class of clinical syndromes defined by spontaneous chromosomal breakage [1]. AT is distinct from the others, however, in that the chromosomal damage observed in the lymphocytes is highly non-random, primarily involving chromosomes 7 and 14, and is frequently characterized by telomeric fusions [2-11]. Due to the nature of the cytogenetic anomalies, AT provides a unique opportunity to examine the structure and mechanism of chromosomal rearrangements in general, as well as to elucidate the relationships between immunodeficiency, lymphoid malignancy, and specific chromosomal alterations. CHARACTERISTIC CHROMOSOMAL REARRANGEMENTS IN AT

Translocations and inversions of chromosomes 7 and 14 (Fig. 1), resulting from the faulty rejoining of breaks at four discrete chromosomal bands (7p14, 7q35, 14q12, 14q32) are regularly observed in the T-lymphocytes of AT individuals [7-13]. These rearrangements are not unique to AT, as they are also the most commonly observed acquired rearrangements in the lymphocytes of phenotypically normal individuals [14-20]. In the general population, translocations between chromosomes 7 and 14 occur in about I of every 2000 lymphocytes examined. In contrast, they are found in roughly 1 of every 60 AT lymphocytes [7]. Similarly, the frequency of inversions of chromosomes 7 or 14 in AT lymphocytes is greater than 50 times that observed in non-AT cells [7]. The paracentric inversion of chromosome 14 is now believed to be the most frequently acquired rearrangement in human lymphocytes [21]. Interestingly, the breakpoints involved in these rearrangements of chromosomes 7 and 14 correspond to the chromosomal bands to which immunologically relevant From the Jules Stein Eye Institute (T. L. K.) and the Departments of Medicine (T. L. K., R. S. S.) and Pathology (R. A. G.), University of California, Los Angeles.

Address reprint requests to: Tracy Kojis, Jules Stein Eye Institute, UCLA, 100 S t e i n Plaza, Los Angeles, CA 90024-7008.

Received April 22, 1991; accepted April 24, 1991.

143 © 1991 Elsevier Science Publishing Co, Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 56:143 156 (1991) 0165-4608/91/$03.50

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T . L . Kojis et al.

B

A

C

:ii ";ype i t(7;14) (q35;q12)

Inv(7)(q14;q35)

Type It t(7;14)(p14;q12)

t(14;14) (q 12;q32)

-ii "w(14) q12;q32)

i

7/c,o {![ ti," 14 (q35;q32)

T'ype IV t(7;14)(p14;q32)

t(7;7}(1;/14 q,35}

Figure 1 T-lymphocyte associated rearrangements (T-LARs) occurring in both normal and AT individuals. (A) Inversions of chromosomes 7 and 14; (B) Translocations between chromosomes 7 and 14; (C) Homologous chromosome translocations.

gene c o m p l e x e s map. The T-cell receptor gamma chain (TCR-7), the T-cell receptor beta chain (TCR-B), the T-cell receptor alpha chain (TCR-a), and the i m m u n o g l o b u l i n h e a v y - c h a i n (IGH) genes are localized to 7p15 [22], 7q35 [23], 14q12 [24], and 14q32 [25], respectively (Fig. 2). The proximity of the four n o n - r a n d o m sites of damage in l y m p h o c y t e s to the map positions of the i m m u n o g l o b u l i n superfamily genes has led to speculation that the characteristic rearrangements observed in AT result from the illegitimate joining of these loci during the normal recombinatorial process necessary for the p r o d u c t i o n of TCR and Ig proteins [26]. This h y p o t h e s i s led to the definition of four additional sites of n o n - r a n d o m damage in AT l y m p h o c y t e s on chromosomes 2 and 22. Chromosomal bands 2p11, 2p12, 22q12, and 22q13.2 are involved in reciprocal exchanges with one another and with the four breakpoints on chromosomes 7 and 14 10 times more frequently than expected by chance in both AT and non-AT lymphocytes [13, 27, 28]. Three of these sites c o r r e s p o n d to the regional localizations of T-cell-specific and i m m u n o g l o b u l i n loci (Fig. 2). The i m m u n o g l o b u l i n light-chain genes, w h i c h are transcriptionally active in B-lymphocytes, map to 2 p l l (IGK) [29] and to 22q12 (IGx) [30, 31]. The Tcell differentiation antigen gene CD8 (T8, Leu2) is localized just distal to the k a p p a light-chain gene at 2p12 [32]. Breakage at the eight sites on chromosomes 2, 7, 14, and 22 generate a set of regularly observed rearrangements in the lymphocytes of AT individuals. However, these characteristic rearrangements do not occur uniformly in all cell types of AT i n d i v i d u a l s [33-35]. The rearrangements of chromosomes 7 and 14, represented in Figure 1, are p r e d o m i n a n t in T-lymphocytes and are notable for their absence from Blymphocytes, bone marrow, and skin fibroblasts [33, 35-38]. These anomalies of c h r o m o s o m e s 7 and 14 will subsequently be referred to as T - l y m p h o c y t e - a s s o c i a t e d rearrangements or T-LARs. I n d i v i d u a l T-LARs demonstrate different propensities for clonality. The translocations and inversions involving chromosome 7 rarely occur in large clonal populations. Usually not more than 1 0 - 1 5 % of the mitoses examined in a given i n d i v i d u a l will bear the same c h r o m o s o m e 7 T-LAR [7, 35], and these tend not to be monoclonal

145

Cytogenetics of Ataxia-Telangiectasia

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2 Figure 2 Map locations of immunologically relevant and cancer susceptibility loci which correspond to T-LAR and B-LAR breakpoints. TCR a, TCR fl, and TCR 7: the T-cell receptor loci. IGH, IG)t, and IGK: the immunoglobulin heavy-chain and the two immunoglobulin light-chain genes, respectively. T8: the GD8 locus.

in origin. Populations of type I 7;14 translocations (Fig. 1) were shown to be polyclonal in two unrelated non-malignant AT patients [9]. There is, however, at least one reported case of a large type III 7;14 translocation (Fig. 1) clone in an AT patient with a T-cell m a l i g n a n c y [39], suggesting that the polyclonal nature of 7;14 translocations is m a s k e d by malignant transformation. Conversely, the c h r o m o s o m e 14 inversions and 14;14 t a n d e m translocations are frequently m o n o c l o n a l in nature, c o m p r i s i n g between 2 and 100% of an AT individual's cells [3, 5, 6, 33, 35, 40-44]. Large clones of either the t(14;14) or the inv(14) can exist for years with no malignant transformation [12, 35, 42, 46]. Again, however, clonal rearrangements of c h r o m o s o m e 14, specifically the t(14;14)(q12;q32), are often associated w i t h T - l y m p h o c y t i c malignancies in AT [33, 42, 45, 46]. Sporadic cases of both the paracentric inverted 14 and the 14;14 t a n d e m translocation have also been observed. However, the clonal and sporadic forms of each may not be precisely identical, having slightly different chromosomal breakpoints. The p r o x i m a l site of breakage is cytogenetically defined as 14q11.2 for clonal chromosome 14 rearrangements and as 14q12 for the sporadic cases [36, 43]. Similarly, the distal b r e a k p o i n t varies in its location: 14q32.109 for clonal and 14q32.3 for sporadic c h r o m o s o m e 14 T-LARs [21, 43]. Molecular breakpoints are discussed below. Rearrangements between c h r o m o s o m e s 2, 22, and 14 are far more c o m m o n in Bl y m p h o c y t e s [34, 37, 47], although they have been observed in a few T-lymphocytes

146

T.L. Kojis et al. [13, 27]. This would be expected if one considers that during B-cell differentiation, somatic rearrangement of TCR gene complexes sometimes occurs as well [48-51]. The B-lymphocyte-associated rearrangements (B-LARs) may have different breakpoints from their T-cell counterparts. In B-lymphocytes, chromosome 22 appears to break at 22q13.2 and chromosome 2 at 2p11, the bands associated with the B-cellactivated immunoglobulin light-chain genes. While in T-cells, the sites of damage on chromosomes 2 and 22 probably occur at 2p12, the map location of the T-cellspecific gene CD8, and at 22q13.2. Analysis of the exact breakpoints is hindered by the very low incidence and sporadic nature of B-LARs. Utilization of polymerase chain reaction (PCR) technology, as has been done for an inv(7) [38, 52], to identify hybrid gene sequences generated by chromosomal translocations and inversions, may facilitate the precise definition of these breakpoints. The cytogenetics of AT bone marrow cells have not been extensively studied. In the few cases analyzed, AT bone marrow cells are cytogenetically normal, demonstrating no tendency for increased chromosomal damage or rearrangement [3, 33, 41]. AT fibroblasts, while exhibiting higher rates of chromosomal damage than either B- or T-lymphocytes [5, 35, 40, 41, 47], do not manifest any of the LARs, nor are they characterized by any consistent chromosomal changes [2, 6, 35, 53, 54]. Furthermore, unlike the lymphocytes, there is no obvious clustering of breakpoints at a subset of chromosomal bands in the fibroblasts [35]. Of further interest, breakpoints near the 11q23 map location of the AT gene(s) [55] have not been observed in any cell type from any AT patient.

MOLECULAR CHARACTERIZATION OF LARs

The correlation of LAR breakpoints with the regional localizations of immunologically relevant genes suggests that T-LARs may be the chromosomal manifestations of interlocus recombination between T-cell receptor and immunoglobulin heavy-chain gene complexes. The first evidence for such recombination was from a malignant non-AT cell line, SUPT-1, derived from a T-cell lymphoma bearing an inv(14) [54, 56-59]. The 14qll breakpoint lies within the joining region of the TCR-a (J~) and the distal breakpoint falls within the variable region of IGH (VH). This site-specific recombination between TCR-a and IGH results in an in-frame transcribed hybrid gene that could potentially be translated into a functional hybrid antigen receptor [54, 56-

59]. Because both the TCR-a and IGH loci are disrupted, the mechanism of rearrangement is postulated to be a recombinase-mediated joining of signal sequences. A T-cell-specific 5.2-kb polyadenylated RNA, normally transcribed from within the IGH locus at 14q32, is found adjacent to 14qll sequences in the inverted 14 of SUPT-1. Germline IGH variable sequences, near this RNA transcription unit, may therefore be available for rearrangement in T-cells and not be strictly limited to B-cell recombination [60]. The hypothesis that a faulty recombinase is responsible for a TCR-IGH hybrid gene is further supported by evidence that TCR and Ig loci utilize a common recombinase for gene assembly and that B- or T-cell specificity is controlled by accessibility of these DNA sequences to the recombinase [61]. The tumorigenicity of SUPT-1 cells may be unrelated to the TCR°IGH recombination. SUPT-1 has additional chromosomal changes including a t(7;9)(q35;q32) translocation, a deletion of the long arm of chromosome 6, and a deletion of the 3' flanking region of c - m y c [62, 63]. The inv(14) may be coincidental to the lymphoma. Breakpoints from two other malignant non-AT lines bearing chromosome 14 TLARs have been characterized. Linv, a T-cell chronic lymphocytic leukemia line carrying an inv(14), and Pt, a T-cell pro-lymphocytic leukemia cell line with a 14-14 tandem translocation, are similar to SUPT-1 only in their 14qll breakpoints. The

Cytogenetics of Ataxia-Telangiectasia

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breaks occur at the 5' ends of joining segments, the normal location for typical and productive V-J recombination within the TCR-a locus [64]. Furthermore, Linv and Pt have heptamer-nanomer recombination signal sequences at their centromeric breakpoint junction, suggesting that an abnormal recombinase is causally related to these T-LARs [65]. Unlike SUPT-1, the 14q32 breakpoints in both Pt and Linv occur centromeric to IGH. Both cell lines retain heptameronanomer signal sequences at their distal breakpoints, but the site of breakage in Pt is not immediately adjacent to them. The Pt 14q32 breakpoint does occur at the 3' end of a sequence with striking homology to immunoglobulin family joining (J) segments [54]. As only TCR-a is involved in both of these ToLARs, neither is the result of TCR-IGH recombination. The first AT clones whose rearrangement breakpoints were molecularly determined are derived from two patients [45, 66] with malignant T-cell leukemic cells bearing 14;14 tandem translocations. In both cases there is site-specific recombination between a region immediately 5' to a J~ segment at 14qll and a region of 14q32 centromeric of IGH [67-69]. A faulty recombinase is again implicated for both. Recombinase signal sequences are immediately adjacent to the breakpoints [68, 69] and there is evidence for N-region addition in at least one case [69]. A malignant T-cell AT clone, AT5B1, bearing an inv(14), yielded ambiguous results concerning the likelihood of recombinase-mediated rearrangement [59]. Like four of the previous five cell lines, AT5B1 has its 14qll breakpoint within J~ and its 14q32 breakpoint centromeric to IGH at 14q32.1. However, the heptamer-nanomer recombinase signal sequences are only present adjacent to the proximal (14q11) break and are completely absent from the distal inversion site [59]. A malignant AT clone bearing a type III (Fig. 1) 7;14 translocation [39] exhibits the same features as the other malignant T-LAR AT clones examined. The J region of TCRofi at 7q35 is joined to the region 14q32.1, which lies approximately 10 megabases centromeric to IGH. Again the translocation occurs immediately 5' of the joining region of a TCR locus, recombinase-like signal sequences are present on both chromosomes 7 and 14 adjacent to the breakpoints and, finally, N-region addition appears to have occurred at the translocation junction [39]. Additionally, there is an inverted duplication of the 14q32 region in this type III 7;14 translocation. This duplication is comparable to the one found in a malignant AT 14;14 tandem translocation clone, characterized by Johnson et al. [67], which encompasses 26 kilobases of the constant-mu region of IGH in addition to some 5' flanking sequences. A similar anomaly may be present in an AT clone, described by Aurias and co-workers bearing a t(14;14) translocation with a cytogenetically defined inverted duplication spanning the region 14q32.1 through 14q32.3 [43, 70]. The significance of such duplications of 14q32 in the malignant cells of AT patients is uncertain. The frequency of these duplications may be underestimated due to the difficulty in detecting them without molecular or very good high-resolution cytogenetic techniques. The only non-malignant AT 14;14 tandem translocation analyzed is derived from a cell line, AT2B1, 70% of whose cells carry the T-LAR [70, 71]. As determined by in situ hybridization, the breakpoints in this non-malignant clone are similar to the malignant t(14;14) T-LARs. The 14qll breakpoint is within the TCR-a and the 14q32 break is proximal to IGH. Non-malignant cells, derived from an AT individual manifesting an inv(7) in 1-5% of his T-cells, demonstrated evidence of site-specific recombination between the variable region of TCR-7 (V~) at 7p14 and a TCR-fi joining region (J~) at 7q35 [72]. This illegitimate joining generated an in-frame hybrid gene, like that of the SUPT-1 line, which was transcribed and could potentially produce hybrid antigen receptors. Thus, the molecular data highlight two types of rearrangements: those involving

148

T.L. Kojis et al. recombination between immunoglobulin superfamily genes and those that juxtapose TCR sequences and a region of 14q32 centromeric to the IGH locus. The former class of rearrangements result in trans-genes whose hybrid V-J junctions are analoguous to those formed within a single locus. Normal recombination signals seem to be utilized, suggesting that both loci are simultaneously accessible to a recombinase. This illegitimate recombination within the immunoglobulin superfamily of genes is not unique to AT as evidenced by the TCR-a/IGH hybrid gene in the SUPT-1 cell line. Sklar et al. [73], using the polymerase chain reaction (PCR), found evidence for translocations between the TCR gamma and delta chain gene complexes in most of the phenotypically normal individuals studied, suggesting that somatic recombination, in general, may be an error-prone process. These studies are now being extended to AT patients. However, these hybrid genes occur significantly more frequently in AT individuals as compared to normal controls. Hybrid TCR-7/TCR-~ genes, as detected by PCR analysis, have an incidence in AT cells about 70 times that observed in normal cells [52]. This elevated frequency of hybrid genes in AT cells is consistent with the published frequencies of T-LARs in AT and normal populations [7]. RNA transcripts from hybrid genes are found in both normal and AT cells. However, unlike the genomic sequences of the hybrid genes, most of the mRNA transcripts maintain open reading frames (~ 80% versus < 50%), implying that there may be enhanced expression of potentially functional hybrid antigen receptors [52]. Theoretically, the hybrid genes could significantly affect the immune response, either positively by increasing the TCR repertoire or negatively by producing ineffective hybrid antigen receptors that result in some degree of immunodeficiency [52]. Alternatively, the hybrid immunoglobulin genes could be the result of sporadic recombinatorial events conferring no proliferative advantage and consequently having little or no effect on the immune response [36, 65]. The second type of molecularly defined T-LARs involves band 14q32.1, a region 15-20 megabases centromeric to IGH; these may play a role in the T-lymphocytic malignancies characteristic of AT. The 14q32.1 region has potential recombination (heptamer-nanomer) signal sequences analogous to those found in IGH [65, 67-69]. Unlike the hybrid immunoglobulin superfamily genes, the TCR-14q32.1 rearrangements seem to confer a proliferative advantage on the cells bearing them, perhaps through the activation of a proto-oncogene [24] or a T-cell-specific growth-affecting locus [53] within 14q32.1. This situation is analogous to the benign clonal expansion of chronic myelogenous leukemia, which is marked by the Philadelphia chromosome (Ph). Just as the Ph seemingly confers a proliferative advantage on the cell lineage carrying it, rearrangements juxtaposing TCR loci with 14q32.1 may similarly predispose the cells to the additional genetic changes ultimately causing blastic transformation. Both types of T-LARs, those involving only immunoglobulin superfamily loci and those associated with sequences at 14q32.1, provide evidence that the AT gene may be related to faulty recombinase activity, as suggested by Bridges and Harnden in 1981 [74]. A defect in a recombinase would explain many of the phenotypic features of AT. An abnormal recombinase could affect DNA repair efficiency and fidelity, resulting in generally increased rates of chromosomal damage and telomeric fusions. The high frequency of spontaneous chromosomal aberrations, which occurs to a lesser extent in phenotypically normal individuals, could be a by-product of accelerated rates of sporadic, but normally occurring, faulty recombination. The immunodeficiency observed in most AT patients might result from an increased frequency of aberrant Ig-TCR hybrid genes, while activation of an oncogene or a growth-promoting gene at 14q32.1 in T-LARs could account for the T-cell malignancies characteristic of AT. It should be noted, however, that the recently cloned recombinases RAG-1 and

149

Cytogenetics of Ataxia-Telangiectasia

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Figure 3 Examples of telomeric fusions in metaphases from the lymphocytes of an AT individual. (a) Two dicentric chromosomes, tf, a 6p;6p telomeric fusion and an 11q;12q telomeric fusion, and a ring chromosome 17, r. (b) A multi-centric telomeric fusion, tf, involving three chromosomes: 9, 11, 19. i inv(14), n normal chromosome 14.

RAG-2 [75] localize not to c h r o m o s o m e 11q23, the site of the AT gene(s), but to the short arm of c h r o m o s o m e 11 (D. Baltimore, personal communication). TELOMERIC FUSIONS

Telomeric fusions, the end-to-end joinings of chromosomes with no apparent loss of material (Fig. 3), are another cytogenetic hallmark of AT [4, 5, 10, 35, 76]. Unlike LARs, telomeric fusions do not regularly occur in the cells of p h e n o t y p i c a l l y normal i n d i v i d u a l s nor in those of the other two spontaneous c h r o m s o m a l breakage syndromes [1]. They are frequently observed in malignant non-AT cells, i n c l u d i n g solid tumors, p r e - T - c e l l acute l y m p h o c y t i c leukemias, and B-cell l y m p h o i d leukemias [77]. Also unlike LARs, w h i c h are tissue specific, telomeric fusions are observed in all cell types, but not necessarily in all AT individuals. A n analysis of seven AT i n d i v i d u a l s [35] revealed that telomeric fusions were prevalent in both l y m p h o c y t e s and fibroblasts (Table 1). Five i n d i v i d u a l s (Group I) lacked clonal LARs in their T - l y m p h o c y t e s while two others (Group II) each had a clone bearing the paracentric inversion of chromosome 14 in 100% of the cells examined. There was a marked difference in the frequency of telomeric fusions in the l y m p h o c y t e s of the two groups. The 2 6 - f o l d greater incidence of telomeric fusions in Group !I m a y be related to the presence of the single large inv(14) clone in their l y m p h o c y t e s . Hecht et al. [3] noted that the frequency of dicentrics was directly related to the p r o p o r t i o n of clonal cells in samples of AT lymphocytes. This between-group difference in the frequency of telomeric fusions was not seen in the fibroblasts (Table 1). Further, the overall rate of telomeric fusions in the fibroblasts was nearly twice that observed in the lymphocytes (0.046 vs. 0.025); this is consistent with the greater degree of chromosomal damage observed in the fibroblasts [35]. Figure 4 presents the distribution of the telomeres involved in the chromosomal fusions observed in both l y m p h o c y t e s and fibroblasts. All the telomeric fusions observed were non-clonal in nature. The s o m e w h a t n o n - r a n d o m distribution of affected telomeres in this study may reflect n o n - r a n d o m breakage w i t h i n i n d i v i d u a l s or s a m p l i n g bias. Previous reports of telomeric fusions in AT suggest that the telo-

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T . L . Kojis et al. Table 1

Group I

Frequency of telomeric fusions per i n d i v i d u a l cell in lymphocytes and fibroblasts. Patient

Lymphocytes

Fibroblasts

JR AR

0 0.008

0.120 0.040

BR

Group II

0

0

CS RS

0.010 0 Average = 0.003

0 O.O8O 0.048

CL TT

0.142 0.014 Average - 0.078

0.040 0.040 0.040

meres i n v o l v e d are r a n d o m l y distributed throughout the genome [1, 4, 10, 11, 77]. Certainly their w i d e s p r e a d occurrence in AT lymphocytes, AT fibroblasts, and tumor cells, in c o n j u n c t i o n with their absence from normal cells, suggests that an understanding of this p h e n o m e n o n may elucidate the underlying defect in AT and its relationship to tumorigenicity. Telomeres stabilize chromosomes. They are comprised of a variable n u m b e r of short t a n d e m repeats with one G-rich and one C-rich strand. The G-rich strand, w h i c h is oriented 5' to 3' toward the chromosome end, overhangs the C-rich strand by 1 2 - 1 6 u n p a i r e d residues and can fold back on itself in vitro through n o n - W a t s o n Crick G - G base pairing [78]. Telomerase, a specialized reverse transcriptase, synthesizes the G-rich strand and is responsible for de novo telomere elongation. The C-rich strand is replicated by semi-conservative mechanisms [78]. There is some evidence, at least in yeast, that telomeric elongation can occur through recombination. The 3' end of one telomere recombines with internal telomeric sequences of another telomere whose translocated repeats are regenerated by polymerase using the existing template [79]. Telomeric fusions, resulting in ring, dicentric, and multicentric chromosomes [Fig. 3), m a y arise in a variety of ways: as a result of telomeric sequence loss, either due to faulty telomerase activity or to telomeric degradation, via intertelomeric recombination, or in the course of normal telomeric replication. The actual m e c h a n i s m of telomeric fusion formation remains obscure as the mode of normal telomeric replication continues to be m u c h debated. Telomerase may not be active in all cell types. Immortal cell lines, such as tumor cells, m a i n t a i n telomere integrity by balancing telomere elongation by telomerase with telomere loss due to semi-conservative DNA replication processes. Conversely, somatic cells may lack telomerase activity or, at least, a renewable source of telomerase, as e v i d e n c e d by the progressive decrease in telomere length with increasing cell age, as defined by increasing numbers of cell divisions, both in vitro [80, 81] and in vivo [81]. One consequence of losing telomeric sequences m a y be an increased i n c i d e n c e of telomeric fusions. If new telomeric repeats cannot be generated, exposed c h r o m o s o m a l ends can be stabilized by fusing to one another. The finite doubling capacity of untransformed m a m m a l i a n cells may be due to loss of telomeric DNA and the eventual deletion of sequences critical to cell survival [80]. Senescent cells exhibit both a high frequency of telomeric fusions [82] and telomeric sequence loss [80]. By analogy, the p r e p o n d e r a n c e of telomeric fusions and the p r e m a t u r e senescence of AT cells may be indicative of accelerated rates of telomeric sequence loss.

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Figure 4 Distribution of telomeres involved in telomeric fusions in the lymphocytes and fibroblasts of AT patients previously studied in reference [35].

Alternatively, if telomeric fusions are a normal intermediate structure in the telomeric replication cycle, as suggested in a more radical model of telomere maintenance [83], their occurrence in somatic cells w o u l d be unrelated to telomeric sequence loss, but rather related to faulty replication mechanisms. Defects in telomere replication processes could p r o d u c e c h r o m o s o m a l l y unbalanced daughter cells, resulting in increased tumorigenicity or cell death, both features of AT cells. Cox et al. [84] tested the fidelity of repair of double-stranded (ds) DNA damage in AT and n o r m a l l y m p h o b l a s t o i d cell lines. While the AT cell line was not i m p a i r e d in its ability to ligate broken strands, the dsDNA breaks were faithfully and accurately repaired in significantly fewer AT cells than in normal controls. Furthermore, deletions and rearrangements of sequences near the breakpoints were more frequently observed in AT cells. The authors propose a d i s e q u i l i b r i u m between DNA ligases and exonucleases, in favor of exonucleases, as the underlying DNA repair defect in AT. However, as has been p r o p o s e d for T-LARs and B-LARs, faulty recombinase activity could be causally related to telomeric fusions. Not enough is k n o w n about telomere replication in somatic cells to draw firm conclusions. Lastly, telomeric fusions m a y arise through t e l o m e r e - t e l o m e r e recombination. Experiments in yeast suggest that telomeric repeats have an increased t e n d e n c y to recombine with one another, possibly as an alternative means of telomere elongation

152

T.L. Kojis et al. [79]. Such a m e c h a n i s m of telomeric fusion formation in AT is consistent with the high rates of non-telomere inter-locus recombination observed in the lymphocytes, and suggests a defect in the processes that govern recombinatorial efficiency and fidelity. Telomere length and sequence maintenance, i n c l u d i n g elongation and reduction, have not been studied in AT cells.

CONCLUSION AT is u n i q u e as a spontaneous chromosomal breakage syndrome in having tissuespecific chromosomal rearrangements and telomeric fusions. T-LARs and B-LARs are the chromosomal manifestations of illegitimate joinings of immunologically relevant and/or cancer-susceptibility-related loci. The molecular definition of LARs suggests a c o m m o n m e c h a n i s m of rearrangement by a faculty recombinase. The m e c h a n i s m of telomeric fusion formation is less clear, but may also involve a faulty recombinase in c o n j u n c t i o n with decreased or absent telomerase activity. A defective recombinase w o u l d provide a satisfying explanation unifying the diverse cytogenetic abnormalities observed in various AT cell types. In turn, the chromosomal changes could be seen as the cytogenetic manifestations of the defective processes u n d e r l y i n g the i m m u n o d e f i c i e n c y , cancer susceptibility, and premature cell senescence typical of AT. Future studies, more clearly defining the AT DNA repair defect, will facilitate a better u n d e r s t a n d i n g of the normal processes of DNA repair and chromosomal integrity. This work was supported, in part, by grants from the U.S. Department of Energy, the AtaxiaTelangiectasia Medical Research Foundation, and the National Cancer Institute. R.A.G. and R~S.S. are members of the Jonsson Cancer Center.

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