21 Monosomy 1P36 As a Model for the Molecular Basis of Terminal Deletions

Chapter 21 / Terminal Deletions of 1p36 301 21 Monosomy 1p36 As a Model for the Molecular Basis of Terminal Deletions

Blake C. Ballif, PhD and Lisa G. Shaffer, PhD

CONTENTS BACKGROUND INTRODUCTION MECHANISMS THAT GENERATE AND STABILIZE TERMINAL DELETIONS GENOMIC ARCHITECTURE AND THE GENERATION OF TERMINAL DELETIONS OF 1P36 SUMMARY REFERENCES

BACKGROUND Deletion of the most distal, telomeric band of human chromosomes can result in a variety of mental retardation and multiple congenital anomaly syndromes. These terminal deletions are some of the most commonly observed structural chromosome abnormalities detected by routine cytogenetic analysis. Terminal deletions of 1p36 occur in approx 1 in 5000 live births, making it the most frequently observed terminal deletion and one of the most commonly observed mental retardation syndromes in humans. Molecular characterization of subjects with monosomy 1p36 indicates that, like other terminal deletions, 1p36 deletions have breakpoints occurring in multiple locations over several megabases and are comprised of terminal truncations, interstitial deletions, complex rearrangements, and derivative chromosomes. In addition, cryptic interrupted inverted duplications have been observed at the end of terminally deleted chromosomes, suggesting premeiotic breakage–fusion–bridge (BFB) cycles can be intermediate steps in the process of generating and stabilizing terminal deletions of 1p36. Overall, these observations are identical to those made in yeast and other model systems in which a double-strand break (DSB) near a telomere can be repaired by a variety of mechanisms to stabilize the end of a broken chromosome. Furthermore, sequence analysis and fluo- rescent in situ hybridization (FISH) mapping of the terminal 10.5 Mb of 1p36 including a variety of terminal deletion breakpoint junctions indicate that segmental duplications, low- copy repeats (LCRs), and short repetitive DNA sequence elements may mediate the generation and stabilization of terminal deletions of 1p36. We hypothesize that nonallelic homologous recombination (NAHR) between palindromic or inverted LCRs in the subtelomeric region of

From: Genomic Disorders: The Genomic Basis of Disease Edited by: J. R. Lupski and P. Stankiewicz © Humana Press, Totowa, NJ 301 302 Part IV / Genomic Rearrangements and Disease Traits

1p36 could generate a dicentric chromosome that is broken at a random location during the subsequent anaphase as the centromeres move to opposite poles. This model suggests that the molecular basis of terminal deletions may be directly linked to genomic architectural features in the subtelomeric regions that generate the initial, variable-sized terminally deleted chromosome, and that stabilization of the broken chromosome occurs by one of a variety of competing DSB repair pathways.

INTRODUCTION Telomere Structure and Function The distal ends of all linear chromosomes are capped by a specialized protein–DNA complex known as the telomere. The term “telomere” was originally put forth by Muller in 1938 to describe the “terminal gene” required for “sealing the end” of a broken chromosome (1). This work was subsequently expanded upon by McClintock (2,3), who demonstrated that broken chromosomes that have lost a telomere form end-to-end fusions resulting in dicentric chromosomes that are inherently unstable. In addition to preventing chromosome fusions, telomeres have now been shown to play critical roles in a number of cellular processes, most notably in the replication of linear chromosome ends (4). Furthermore, telomere associations during meiosis suggest that they may be involved in homolog pairing and recombination and that they are necessary for faithful segregation of the chromosomes (5). Telomeric DNA consists of tandem repeats of simple G-rich sequences that show remark- able conservation throughout eukaryotic evolution. All human chromosomes terminate with approx 2–20 kb of the simple tandem repeat (TTAGGG)n (6). Proximal to this telomeric repeat tract is a structurally complex region of subtelomeric DNA that can extend several hundred kilobases from the end of most chromosomes and has been shown to be highly polymorphic (7–10). These subtelomeric regions are enriched with segmental duplications interspersed with interstitial (TTAGGG)n-like sequences and other subtelomeric repeat sequence blocks that can be shared by a number of different chromosome ends (10). In addition, early chromosome banding (G-banding) studies indicated that the telomeric regions of most human chromosomes stain G-negative and contain GC-rich isochores and are, therefore, thought to be regions that contain abundant genes (11,12). Thus, telomeric deletions and/or rearrangements have been predicted to result in more significant and serious phenotypic abnormalities than similar-sized deletions and/or rearrangements at other chromosomal locations (11,13,14). Analysis of the draft sequence of the Human Genome assembly has recently confirmed that telomeric regions are gene-rich, although not to the extent originally estimated (10). Telomeric Aberrations and Mental Retardation Mental retardation affects approx 3% of the world’s population (15). Although chromosomal and genetic disorders are thought to account for 30–40% of all cases with an additional 10–30% accounted for by environmental factors, the cause of mental retardation in the vast majority of subjects is still unknown (16–18). It has been estimated that approx 20% of mental retardation is caused by a cytogenetic abnormality, with aneuploidy, mainly trisomy 21, being the most common. Although estimating the second most common cytogenetic cause may have some biases, in recent years telomeric abnormalities have emerged as a major yet previously under-recognized cause of mental retardation in the population (19). Chapter 21 / Terminal Deletions of 1p36 303

In the past decade, numerous submicroscopic chromosomal abnormalities in subjects with apparently normal karyotypes and mental retardation or multiple congenital anomalies have been identified by molecular methods such as DNA polymorphism analysis (20–22), FISH (13,23,24), and microarray-based comparative genomic hybridization (array CGH) (25,26). Although it is difficult to compare directly these various studies because of differences in patient selection criteria and variations in the subtelomeric screening methodologies, the combined data suggest that approx 5% of subjects with idiopathic mental retardation or dysmorphic features and apparently normal karyotypes may have submicroscopic deletions and/or cryptic rearrangements at the telomeres (19,27). Terminal Deletions and Monosomy 1p36 Telomeric regions of the genome are of particular interest in clinical cytogenetics because, owing to their light G-negative staining on G-banded chromosomes, rearrangements of these regions are difficult to identify by conventional cytogenetic methodology. Nonetheless, terminal deletions are one of the most commonly observed structural chromosomal abnormalities detected by routine chromosome analysis. The first deletion identified in humans was the terminal deletion of 5p associated with Cri- du-chat syndrome (5p-) (28). Shortly thereafter, terminal deletions of 4p were identified and the resulting syndrome, Wolf-Hirschhorn, was described (29,30). With the advent of chromosome banding techniques and the more recent molecular cytogenetic technology, visible or subtle terminal deletions have been identified for every human chromosome (14). This has allowed the delineation of a number of mental retardation syndromes associated with deletion of the telomeric bands of the chromosomes including Miller-Dieker (17p-) (31) and monosomy 1p36 (1p-) (32). However, the molecular mechanism(s) that generates and stabilizes terminal deletions is only beginning to be elucidated. Terminal deletions of 1p36 occur in approx 1 in 5000 live births, making it the most frequently observed terminal deletion and one of the most commonly occurring mental retardation syndromes in humans (27,33). Monosomy 1p36 is characterized by distinct facial features including deep-set eyes, flat nasal bridge, asymmetric ears, and pointed chin. Other features include mild to severe mental retardation, seizures, hearing impairment, vision disorders, growth failure, hypothyroidism, oropharyngeal dysphagia, orofacial clefting, and cardiac defects (34,35). More than 100 subjects with deletions of 1p36 have now been systematically analyzed (34 and unpublished data). Although the majority of cases of monosomy 1p36 have been identified through cytogenetic analysis, this deletion is difficult to visualize by routine chromosome analysis at the 400–550 band resolution. However, the majority (98%) of terminal deletions of 1p36 can be visualized retrospectively using good quality chromosomes at a resolution of 550 bands or greater, with careful attention to chromosome band 1p36 (27). Nevertheless, nearly half of patients studied had at least one chromosome analysis, some- times two, interpreted as normal (27). Furthermore, the size and complexity of the deletion cannot be distinguished at the resolution of the light microscope. A variety of molecular cytogenetic techniques have revealed that deletion sizes in monosomy 1p36 vary widely with no single common breakpoint (32,34,36). Additionally, terminal deletions, interstitial deletions, complex rearrangements, and derivative chromosomes have been identified among the study cell lines (34,37,38). Given that 1p36 deletions are relatively common and, like other terminal deletions, comprise a heterogeneous group of submicroscopic rearrangements 304 Part IV / Genomic Rearrangements and Disease Traits with breakpoints occurring in multiple locations over several megabases, monosomy 1p36 has proven to be a valuable model for investigating the molecular basis of terminal deletions.

MECHANISMS THAT GENERATE AND STABILIZE TERMINAL DELETIONS Telomeres provide structural stability to the ends of all linear chromosomes. Loss of a telomere, by either a DNA DSB or by improper telomere maintenance can, if not properly repaired, lead to apoptosis, cell senescence, or severe genomic instability characterized by gene amplifications and deletions as a result of perpetual BFB cycles (4,39). It is now clear that there are at least two general repair pathways that exist within the cell whereby a terminally deleted chromosome can acquire a new telomeric cap. The first pathway is known as “telomere healing” and results in the stabilization of terminal deletions by the direct addition of telomeric repeats onto the end of a broken chromosome (40). The results of telomere healing events are pure terminal truncations. Although true telomere healing events may occur by telomerase-mediated, de novo addition of telomeric repeats on the end of a chromosome (37,40–43), pure terminal truncations can also result from telomerase- independent, recombination-based telomere capture mechanisms from a pre-existing telomere (44,45). The second general pathway has been termed “telomere capture” and refers to the process through which a terminally deleted chromosome acquires a new telomeric sequence from another chromosomal location (46–48). Telomere capture events could potentially occur between sister chromatids, homologs, or nonhomologous chromosome ends and result in derivative chromosomes. Although a number of examples of what appear to be telomere healing and telomere capture events have now been described in a variety of model organisms (37,42,49–54), relatively few cases have been reported for constitutional chromosome abnormalities in humans. Moreover, the precise mechanisms that mediate these pathways in humans are not understood. Furthermore, it is not clear why terminal deletions are so common and which mechanism of repair and stabilization is used most frequently. Molecular studies on a large cohort of subjects with cytogenetically defined terminal deletions of 1p36 using microsatellite analysis, FISH, and array CGH indicate that deletion sizes range from approx 1 Mb to more than 10.5 Mb with the majority (40%) of breakpoints clus- tering between 3 and 5 Mb from the telomere (26,34). Furthermore, telomere FISH studies have identified interstitial deletions (6%), derivative chromosomes (17%), complex rearrangements (5%), and apparently pure terminal deletions (72%) (34,38). Ten rearrangements of 1p36 have been examined at the DNA sequence level (38,55,56) and an additional two were examined by microarray analysis and FISH (57). Based on the previous molecular analyses, seven were apparently pure terminal deletions, three were known derivative chromosomes 1 [der(1)t(1;1)(p36;q44)], and two were apparent duplications of 1p36. Analysis of the breakpoint junctions in these subjects uncovered evidence for BFB cycles, confirmed a telomere healing event, and clarified the mechanisms of telomere capture involved in terminal deletions of 1p36. In the next sections we discuss in detail the various mechanisms by which terminal deletions can be generated and stabilized, most of which have already been observed in terminal deletions of 1p36, and suggest that terminal deletions of 1p36 are a good model for understanding the breakage and stabilization of human chromosomes. Chapter 21 / Terminal Deletions of 1p36 305

Breakage–Fusion–Bridge Cycles Breakpoint junction sequences from two subjects with monosomy 1p36 identified terminal deletions associated with cryptic interrupted inverted duplications at the end of the chromosome (37). These breakpoint sequences are identical in structure to those found in embryonic stem (ES) cell lines and tumor cell lines that have gone through BFB cycles as a result of uncapped sister chromatids being fused by nonhomologous end joining (58,59). The analysis of two additional subjects with apparent duplications of 1p36 showed terminal deletions, duplications, and triplications (57). The mechanisms postulated to result in these complex rearrangements involve BFB cycles (57). These data represent the first evidence of a BFB cycle generating a constitutional chromosome rearrangement resulting in a human genetic disease. Furthermore, this suggests that the observed variability in size of terminal deletions of 1p36 (and other terminal deletions) may be the result of random breaks generated when a fused, dicentric chromosome is broken during anaphase separation of the centromeres. Perhaps most significant is that the identification of BFB cycles suggests that at least some terminal deletions of 1p36 are not generated and stabilized in a single step but pass through an intermediate phase as a broken chromosome that must then be stabilized by some other cellular mechanism. Telomere Healing Pure terminal truncations have been reported in at least 12 terminal deletion cases from a variety of human chromosome ends (37 40,41,43,60–62). Recently, sequence analysis of the precise breakpoint junctions in subjects with monosomy 1p36 confirmed at least one case to be a pure terminal truncation stabilized by telomeric repeat sequences (37). Telomere healing has also been identified in human tumor cell lines (58), mouse ES cells (42), and a number of other model organisms (49,50,52,53). Although it is presumed that pure terminal truncations were stabilized by de novo telomere healing by telomerase, broken chromosomes stabilized by acquisition of telomeric sequences from another chromosome end could appear virtually identical to ones stabilized by de novo telomere synthesis. However, the reported findings of pure (TTAGGG)n telomeric repeats at the sites of healing, as opposed to the variant telomeric repeats that are commonly found in the proximal telomeric region of existing telomeres (37,40,41,43,60,62–64), support de novo synthesis of telomere repeats by telomerase. Even though the lack of variant repeats is consistent with de novo addition, it is not definitive because greater than half of an existing telomere’s 2–20 kb sequence consists of pure telomeric repeats (63,64). In support of de novo synthesis, using an in vitro assay system, human telomerase was shown to preferentially recognize the sequence at the breakpoint of one 16p terminal truncation and to add (TTAGGG)n telomeric repeat sequences (65). Telomere Capture In the past few years, a variety of cryptic telomeric aberrations have been identified, making it clear that many apparently pure terminal deletions, viewed at the resolution of the light microscope, are in fact terminal deletions/broken chromosomes stabilized by telomere capture events (34,36,48,66,67). At least four models have been proposed for the molecular basis of telomere capture. First is the mechanism of NAHR between LCRs in nonhomologous telomeric regions (68), which would predict the simultaneous generation and stabilization of a terminal deletion as a derivative chromosome. Analysis of der(1)t(1;1)(p36;q44) chromosomes from 306 Part IV / Genomic Rearrangements and Disease Traits three subjects with monosomy 1p36 revealed variability in the breakpoint locations without any evidence for homologous sequences or LCRs between the 1p36 deletion and 1q translocation breakpoints (56). This suggests that NAHR did not play a role in mediating these particular rearrangements. However, this does not preclude LCRs within the subtelomeric region of 1p36 from being involved in generating and/or stabilizing terminal deletions by other mechanisms such as subtelomeric pairing with proximal recombination. The second major mechanism for telomere capture, which has been termed “subtelomeric pairing with proximal recombination” (SPPR), suggests that LCRs located within the complex subtelomeric regions can facilitate mispairing of nonhomologous chromosome ends (69,70). A more proximal recombination event between the mispaired chromosomes at one of the more abundant and much shorter repetitive DNA sequence elements could generate a terminal deletion and stabilize it as a derivative chromosome in a single step. Although data from the three der(1)t(1;1)(p36;q44) chromosomes is consistent with a SPPR model in that no LCRs were identified at the breakpoints, microsatellite data from these subjects suggest that these telomere capture events were mediated by intrachromosomal rearrangements. Therefore, to remain consistent with a SPPR model, in these three der(1) cases 1qter sequences must have been translocated to 1pter from a sister chromatid (56). A third mechanism has recently been proposed for generating terminally deleted chromosomes that are stabilized by telomeric sequences originating from the opposite end of the chromosome, as seen in the three der(1)t(1;1)(p36;q44) chromosomes (71). This model pro- poses that a premeiotic exchange between chromosome 1 homologs could result in the “transposition” of 1pter and 1qter sequences. If normal homolog pairing in meiosis occurred followed by a recombination event in 1p36 proximal to the site of interchromosomal “transposition,” then the result could be both a deleted 1p, duplicated 1q chromosome and a duplicated 1p, deleted 1q chromosome (56,71). Although technically possible, this mechanism does not account for the wide range of derivative chromosomes seen in monosomy 1p36 and in terminal rearrangements of other chromosomes. The fourth, and most likely, model for telomere capture suggests that telomeric sequences from another chromosome end could be copied directly onto the end of a broken chromosome through a process known as break-induced replication (BIR) (54). BIR has been studied in yeast systems and it has been recently shown to play a significant role in DSB repair and preventing gross chromosome instability by maintaining telomere integrity (46,51,54,72,73). BIR is a very appealing mechanism in that a broken chromosome could theoretically be healed by copying telomeric DNA from any chromosome end. This could account for the wide range of derivative chromosomes seen in monosomy 1p36, as well as those in other terminal deletion syndromes, in addition to the lack of LCRs at or near the deletion/translocation breakpoints (56). Terminal Deletions of 1p36 As a Model for Mechanisms That Stabilize Broken Chromosomes As a whole, the molecular analysis of terminal deletions of 1p36 is consistent with a number of recent reports that suggest a chromosome broken near the telomere can be stabilized by a variety of competing DSB repair pathways to provide the necessary telomeric cap (54). In particular, telomere dysfunction because of improper telomere maintenance, chromosome breakage, or mutations in various DSB repair and/or S-phase checkpoint pathways results in severe genomic instability typical of cancer (59,61,74–80). This genomic instability is characterized by chromosome fusions and BFB cycles that generate inverted duplications, terminal Chapter 21 / Terminal Deletions of 1p36 307 deletions, and nonreciprocal translocations that are stabilized by several telomere healing and telomere capture mechanisms (54). The recent studies by Kolodner et al. (54) of yeast are of particular interest because they suggest extensive and redundant DNA repair pathways function to keep genomic rearrangements at very low levels. Their data indicate that disruption of the S-phase checkpoints, com- pared to disruption of the G1, G2, or mitotic spindle checkpoints, significantly increases the rate of genome rearrangements (54). These results indicate that replication errors play an important role in generating genomic instability. In addition, all of the genome rearrangements observed following inactivation of the S-phase checkpoint were terminal deletions stabilized by telomeric repeat sequences (54). Kolodner et al. (54) have also shown that mutations in yeast that disrupt the BIR repair pathway, and not the classical DSB repair by homologous recombination, result in a significant increase in genome instability. Furthermore, telomerase-defective yeast show a dramatic increase in the number of BIR-induced nonreciprocal translocations and nonhomologous end-joining chromosome fusions (54). We propose that the diversity in rearrangements observed in terminal deletions of 1p36 illus- trates a similar set of circumstances in human terminal deletions in that they reflect the various DSB repair pathways that were taken to stabilize a broken chromosome. Figure 1 shows a schematic representation of some of the known mechanistic pathways whereby a broken chromosome can be stabilized. Processing a broken chromosome through any one of these pathways can result in a terminal deletion coupled with a subtle telomeric rearrangement—all of which have been observed in monosomy 1p36 subjects (34,37,55,56,81). Furthermore, these observations on terminal deletions of 1p36 suggest that the same variety of subtle telomeric rearrangements observed in other terminal deletion syndromes (60,66,67,82) are also likely to be the result of various DNA repair pathways stabilizing broken chromosomes.

GENOMIC ARCHITECTURE AND THE GENERATION OF TERMINAL DELETIONS OF 1P36 Repetitive DNA Sequence Elements In recent years, numerous examples in the literature have documented repetitive DNA sequence elements as promoters of aberrant homologous recombination and DNA DSBs (83,84). This is evidenced by the vast number of cases in which repetitive DNA sequence elements are found at the breakpoints in constitutional chromosomal aberrations that cause genetic disease and in sporadic chromosomal abnormalities that cause cancer (83,84). Presumably, aberrant homologous recombination between closely related repetitive DNA sequence elements (such as Alu and L1 sequences) can mediate these rearrangements without a prolonged period as a broken chromosome. In four subjects with apparently terminal deletions of 1p36, the breakpoints occurred in repetitive DNA sequence elements (55). This suggests that aberrant homologous recombination may have been involved in generating and/or stabilizing these terminal deletions. Aberrant homologous recombination between Alu elements has also been attributed to the generation of two subtle interstitial deletions of 16p that were initially thought to be simple chromosomal truncations (60,85). A similar situation has also been reported for a terminal deletion of 18q in which satellite III DNA sequences were located at the breakpoint (86). Some repetitive DNA sequence elements have also been shown to be susceptible to genomic rearrangements including deletions (83,84). Microsatellite, minisatellite, and other short 308 Part IV / Genomic Rearrangements and Disease Traits

Fig. 1. Mechanisms for stabilizing a broken chromosome. (A) Premeiotic or meiotic DNA damage generates a double-strand break that converts an intact normal chromosome into a broken, terminally deleted chromosome. Although DNA repair pathways could function to restore an intact normal chromosome (B), cell-cycle checkpoints may recognize the damaged chromosome and target the cell for an apoptotic pathway (C). Thus, neither of these two possible pathways (B or C) would be observed in subjects with monosomy 1p36 because a restored normal chromosome (B) would not produce a phenotype and an apoptotic cell (C) would simply not survive. However, a variety of competing DNA repair pathways could function to stabilize a broken chromosome. Terminal truncations could be formed by telomerase-mediated de novo addition of telomeric repeats or through telomerase-independent mechanisms such as break-induced replication (BIR) (D). (E) Stabilization could also occur by telomere capture forming a derivative chromosome through BIR. If more than one broken chromosome is present in the cell, single-strand annealing or nonhomologous end-joining (NHEJ) could also generate a nonreciprocal translocation. If the original telomere were captured, a true interstitial deletion could also be formed (F). Alternatively, DNA replication and NHEJ could occur (G), resulting in breakage–fusion–bridge cycles that generate inverted duplications and terminal deletions that in turn require stabilization (H). All of these pathways have been proposed in terminal deletions of 1p36. In addition, genomic architectural features, such as palindromic low-copy repeats in the subtelomeric region of 1p36, could result in nonallelic homologous recombination, creating a dicentric chromosome that breaks at a random location during anaphase (I). These types of random breaks could be a source of variable-sized broken chromosomes that are then stabilized by one of several competing double-strand break repair pathways. A similar set of DNA repair pathways that safeguard against genomic instability has been described in yeast systems. (Adapted from ref. 54.)

repetitive sequences are particularly susceptible to replication errors and can generate palindromic, hairpin structures that are prone to cleavage, resulting in DSBs and other chromosomal rearrangements (87–90). Because the breakpoints of four subjects with terminal deletions of 1p36 were shown to be located within repetitive DNA sequence elements, it is reasonable to Chapter 21 / Terminal Deletions of 1p36 309 suggest that some terminal deletions of 1p36 may have been generated by DNA sequences that were susceptible to DNA DSBs (55). These DSBs generate broken chromosomes that must then become stabilized by telomere healing or capture mechanisms to avoid potential BFB cycles. However, repetitive DNA sequence elements found at the end of a broken chromosome may also constitute a favorable site for stabilization by DNA repair mechanisms (54). Low-Copy Repeats and Segmental Duplications What generates the initial broken chromosome and why terminal deletions of 1p36 are ascertained so frequently is still unknown. This could reflect higher instability in this particular region of the genome or increased survival of terminal deletions of 1p36 over other telomeric abnormalities. Interestingly, one method uses an inverted duplication of the short arm (2). This was how McClintock originally formed a dicentric chromosome to generate broken chromosome sand initiate BFB cycles using a maize chromosome 9. A crossover between the normal homolog and the inverted portion of the duplicated chromosome, or between duplicated regions of sister chromatids, resulted in a dicentric chromosome that formed a bridge in anaphase with subsequent breakage (2). Large-scale sequence analysis of the terminal 10.5 Mb of 1p36 indicates that segmental duplications along with palindromic and inverted LCRs may be present in the distal subtelomeric regions (91). The region surrounding these LCRs forms a natural division between the interstitial deletion breakpoints of 1p36 and nearly all of the terminal deletion breakpoints (Ballif, Gajecka, and Shaffer, unpublished data). It is tempting to speculate that NAHR between palindromic or inverted LCRs in the subtelomeric region of 1p36 could be responsible for generating a dicentric chromosome that is broken at a random location during the subsequent anaphase as the centromeres move to opposite poles (Fig. 2). Similar models have been proposed for generating inverted duplication/deficiency chromosomes, although those models were based on subtelomeric LCRs located in inverted orienta- tions and positioned several megabases apart (92–94). The palindromic repeats proposed in this model for terminal deletions of 1p36 would provide a single site for NAHR with subsequent breakage of the newly formed dicentric chromosome generating variable-sized terminally deleted and inverted duplication/deficiency chromosomes. Stabilization of the broken chromosomes could then occur by a variety of DSB repair pathways (Fig. 1). Although duplication/deletion chromosomes would also be expected to occur, it is likely that these reciprocal products have not been ascertained in studies of monosomy 1p36 because they would be predicted to have much smaller imbalances and may present with a mild phenotype that is not typical of the syndrome. Because the genomic sequence of 1p36 is not in its finished form, the precise size and orientation of these putative LCRs is still under investigation (91). However, regions of the genome that contain large duplications are notoriously difficult to map and sequence (68,95,96). A closer examination of the subtelomeric regions of all chromosomes may be useful for understanding the molecular basis of other terminally deleted chromosomes.

SUMMARY Combined, the data presented suggest that: (1) genomic architectural features may ulti- mately play a role in the generation of terminal deletions of 1p36 by generating the double- strand DNA breaks near the telomere, and (2) a variety of DNA repair pathways are responsible for stabilizing the broken chromosomes. Furthermore, based on the variety of rearrangements and the various mechanisms that generate and stabilize broken chromosomes ends, these 310 Part IV / Genomic Rearrangements and Disease Traits

Fig. 2. Model for nonallelic homologous recombination (NAHR) between palindromic low-copy repeats (LCRs) in the subtelomeric region of 1p36. Palindromic LCRs are shown as arrowheads. Telomeric repeat sequences are shown as filled circles at the ends of the chromosomes. Unique sequences, shown as solid, hatched, and open boxes, are included for illustrative purposes. (A) Palindromic LCRs can promote misalignment of homologous chromosomes. (B) NAHR between misaligned palindromic LCRs results in the formation of a dicentric chromosome. (C) In anaphase, when the centromeres move to opposite poles, the resulting dicentric chromosome forms a bridge that will eventually break at a random location. (D) Random breakage results in one chromosome with an inverted duplication and a distal deletion and a second chromosome (E) with a terminal deletion. This random breakage could account for the variability in deletion size observed in terminal deletions of 1p36. However, these broken chromosomes must acquire a telomeric cap to be stabilized.

studies indicate that monosomy 1p36 provides a fundamental model for the molecular basis of constitutional terminal deletions in humans. In the future, the delineation of the structure of terminal deletions for other chromosome ends will determine whether the monosomy 1p36 model is representative of the molecular basis of all terminal deletions.

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