Several common syndromes, such as monosomy 1p36 (OMIM 607872), Wolf-Hirschhorn (OMIM 194490), Cri-du Chat (OMIM 123450), monosomy 9p (OMIM 158170), 9q34.3 microdeletions (OMIM 610253), Jacobsen (OMIM 147791), Miller–Dieker (OMIM 247200) and 22q13.3 (OMIM 606232) are associated with terminal subtelomeric deletions of chromosomes 1p, 4p, 5p, 9p, 9q, 11q, 17p and 22q, respectively. With the widespread application of high-resolution genomic analyses, a substantial number of rearrangements involving the subtelomeric regions of all chromosomes have been reported to cause birth defects and mental retardation (12
). Many of these deletions are not mediated by low-copy repeats (LCRs), are of different size and genomic content in each patient, commonly cannot be resolved by conventional cytogenetic techniques and thus might be classified as nonrecurrent rearrangements associated with genomic disorders (15
). Although a characteristic phenotype has been recognized for a few of these rearrangements, little is known about the contribution to the phenotype potentially made by either dosage-sensitive genes or regulatory elements within the deleted region. Furthermore, there is a paucity of information available regarding either molecular recombination mechanisms leading to terminal deletions or genome architectural features potentially causing susceptibility to genomic instability.
Submicroscopic deletion del(9)(q34.3) is a relatively newly described genomic disorder that affects fetal development and results in mental retardation and multiple congenital anomalies. Microdeletions of the 9q34.3 region, like other terminal deletions, have breakpoints occurring in multiple sites of the distal chromosome end. Molecular analysis of patients with monosomy 1p36 (16
) or 9p21–p24 (16
) demonstrated that deletions vary widely up to 20 Mb in size with no single common breakpoint. In patients with monosomy 1p36, 40% of all breakpoints were located 3.0–5.0 Mb away from the telomere (16
), so >50% of these rearrangements can be detected by chromosome analysis. In contrast to monosomy 1p36, and 9p syndromes, deletions involving the 9q34.3 region do not exceed 3.5–4 Mb in size; therefore, such rearrangements are usually beyond the resolution of conventional cytogenetic techniques.
In our study, ~70% of 9q34.3 breakpoints occurred within the most distal 1.2 Mb region (Fig. ). Our studies demonstrate that rearrangements of the 9q34.3 region can be either paternal or maternal in origin. Paternally derived rearrangements were usually de novo
simple terminal deletions (65% of patients), whereas interstitial deletions, complex rearrangements and unbalanced translocations were frequently maternal in origin. Moreover, the majority of small 9q34.3 deletions (<1.2 Mb) were paternal, whereas deletions >1.2 Mb in size were predominantly maternal in origin. In contrast, 60% of patients with monosomy 1p36 had small deletions on the maternally derived chromosome, whereas paternal deletions were larger. Interestingly, the distal 9q34.3 region has significant differences in recombination rates between maternal and paternal chromosomes (18
). Within the segment located 3 Mb away from the 9q telomere, female- and male-specific recombination rates were calculated as 3.5 and 3.7 cM/Mb, respectively, whereas in the most distal 1 Mb region, recombination events in females were not detected at all, and in males, the recombination rate was found to be 1.8 cM/Mb. For the 1p36 region, the peaks of recombination are observed within segments located 4–5 and 5–6 Mb away from the telomere in females and males, respectively (18
). It is possible that location and parental origin of DNA breaks correlates with the recombination map. Thus, nonrecurrent breakpoints observed in patients with subtelomere rearrangements may not be entirely random, but rather more common within specific regions or sequences of the human genome.
The ends of chromosomes are evolutionarily active with a very high rate of meiotic recombination and double-strand breaks (DSBs). Elevated recombination rates within the subtelomeric regions may depend on GC content, density or content of genes, cis
- and trans
-genetic modifiers, chromatin structure or may be associated with specific genomic sequence features. Specific genomic architectural features such as LCRs and short-interspersed repetitive elements (e.g. Alu
) are known to mediate recurrent rearrangements including deletions, duplications, inversions and translocations via NAHR (20
). Such sequences may stimulate, but do not mediate, rearrangements due to NHEJ and FoSTeS (21
). NHEJ has been implicated in pathogenesis of subtelomeric nonrecurrent rearrangements as well (22
). From our junction analysis, we conclude that interstitial deletions in three patients were consistent with NHEJ repair.
The presence of short repetitive elements has been proposed to play a role in generating or stabilizing the terminal deletions; however, it is unclear whether these repeats participate in a recombination events or are involved in DNA replication and repair. In this study, we have determined 43 breakpoint junctions within the subtelomeric 9q34.3 region. Repetitive sequences such as Alu
, LINE, SINE, LTRs and STRs were commonly present at or near the breakpoints (Table ). These repetitive elements are susceptible to DSBs due to replication errors or by formation of unusual DNA secondary structures including cruciforms, hairpins, triplexes, tetraplexes and so on. (24
). In addition, secondary structure can inhibit DNA polymerization or accumulate ssDNA in the replication fork, thus increasing the probability of rearrangements.
A DNA replication model termed FoSTeS has been proposed to explain the complex rearrangements associated with Pelizaeus–Merzbacher disease (OMIM 312080) (11
). This long-distance template-switching replication mechanism has been postulated for Escherichia coli
gene amplification (25
) and for both disease-associated rearrangements and CNVs in the human genome (26
). The microhomology mediated break-induced replication (MMBIR) model represents a more generalized mechanism with specific molecular details and applicable to all life forms. In the FoSTeS/MMBIR model, a DNA replication fork stalls or pauses at DNA lesions, leading to fork collapse and the generation of a single ended double strand DNA, and subsequent restoration of replication by switching to an alternative template using microhomology to prime DNA synthesis on the switched template. Among patients reported in this study, two subjects were found with complex 9q34 rearrangements. Rearrangement in P6 is suggestive of a multistep healing event. Interestingly, a patient with a complex rearrangement including deletion, inverted duplication and triplication of the distal 9q34.3 region has been reported recently (28
). These findings provide additional evidence that diverse mechanisms may be involved in generating subtelomeric chromosomal rearrangements. The deletion segment in P29 was interrupted by a small nondeleted segment. Similarly, multiple deletions were reported previously in three patients with monosomy 1p36 syndrome (16
). Analysis of a junction fragment in P29 revealed a 3 bp microhomology. Rearrangements resulting in complex multiple deletions may be explained by coincidence of independent DSBs events healed by NHEJ mechanism, although the frequency of such aberrations is likely to be extremely low. Alternatively, interrupted deletions and more complex rearrangements can be more parsimoniously explained by the FoSTeS/MMBIR mechanism.
In this study, 50% of patients were detected with simple terminal deletions. In the absence of functional telomeres, eukaryotic chromosomes undergo end-fusion and degradation events, making them generally unstable. At least three mechanisms for healing of terminal deletions have been proposed: de novo
telomere addition mediated by telomerase, capture of an existing telomere resulting in derivative chromosomes and stabilization by BFB cycles, consistent with interrupted terminal deletion and more proximal inverted interrupted duplication rearrangement. Each of these mechanisms has been identified by characterization of terminal deletions (28
) and also is revealed in the present study.
In the reported cases, the truncated chromosomes have been healed by the direct addition of T2AG3 repeats onto nontelomeric sequences. Despite high-resolution array-CGH analysis for the majority of patients with terminal deletions, attempted amplification of the junction fragments was nonproductive. Molecular studies employing a series of probes for FISH or array-CGH analyses showed that many terminal deletions are more complex than anticipated. In our PCR design, we hypothesized healing by the T2AG3 repeats, but not TAR sequences for telomere swaps; the latter may not be spanned by a long PCR designed to amplify the junction. However, some of the terminal abnormalities may be interstitial rearrangements with a distal breakpoint within TAR repeats or be complex as well.
In contrast to recurrent rearrangements that are often associated with genomic architectural features, breakpoints involving the subtelomeric region are highly variable. Molecular studies identified three regions of breakpoint grouping within the 9q34.3 region. Interestingly, unique breakpoints have been reported for most subjects with nonrecurrent deletions involving the 22q13.3 region, but a recurrent breakpoint within a small region of the SHANK3
gene has been identified in three individuals (33
). The loci of breakpoint grouping may represent hot spots with a specific DNA sequence feature or a secondary structure that stabilizes broken chromosome or assist in DNA DSB repair or loading of the telomerase enzyme.
This study shows that multiple mechanisms associated with subtelomeric DNA alterations and repair can be involved in the pathogenesis of the 9q34.3 microdeletion syndrome. The observed mechanisms can also be implicated in rearrangements of other subtelomeric regions; some of these models may be more prevalent depending on chromosome region-specific characteristics, such as genomic architecture, repetitive sequence density or other genomic features. More investigations are required to further understand the mechanisms and frequencies with which they are involved in subtelomeric rearrangements.