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Published online before print
March 9, 2007, 10.1101/gr.5986507 Genome Res. 17:482-491, 2007 ©2007 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/07 $5.00
Letter Low copy repeats mediate distal chromosome 22q11.2 deletions: Sequence analysis predicts breakpoint mechanisms1 Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA; 2 Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA; 3 Children’s Hospital of Minnesota and University of Minnesota, Minneapolis, Minnesota 55455, USA; 4 Departments of Comparative Medicine, Genetics and Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Genomic disorders contribute significantly to genetic disease and, as detection methods improve, greater numbers are being defined. Paralogous low copy repeats (LCRs) mediate many of the chromosomal rearrangements that underlie these disorders, predisposing chromosomes to recombination errors. Deletions of proximal 22q11.2 comprise the most frequently occurring microdeletion syndrome, DiGeorge/Velocardiofacial syndrome (DGS/VCFS), in which most breakpoints have been localized to a 3 Mb region containing four large LCRs. Immediately distal to this region, there are another four related but smaller LCRs that have not been characterized extensively. We used paralog-specific primers and long-range PCR to clone, sequence, and examine the distal deletion breakpoints from two patients with de novo deletions mapping to these distal LCRs. Our results present definitive evidence of the direct involvement of LCRs in 22q11 deletions and map both breakpoints to the BCRL module, common to most 22q11 LCRs, suggesting a potential region for LCR-mediated rearrangement both in the distal LCRs and in the DGS interval. These are the first reported cases of distal 22q11 deletions in which breakpoints have been characterized at the nucleotide level within LCRs, confirming that distal 22q11 LCRs can and do mediate rearrangements leading to genomic disorders.
Chromosome 22q11 shows a high frequency of de novo genomic rearrangement. This instability is attributed to the presence of several large paralogous low copy repeats (LCRs) or segmental duplications (SDs), each containing a complex modular structure and a high degree of sequence identity (>96%) over large stretches of the repeat (Shaikh et al. 2000  ). The LCRs apparently mediate aberrant interchromosomal exchanges during meiosis (Saitta et al. 2004), and 22q11 deletions, which occur in up to 1:4000 live births (Burn and Goodship 1996), are among the most frequent constitutional rearrangements. Other chromosomes are also known to contain similar "rearrangement-promoting" low copy repeats that are implicated in mediating genomic disorders. Examples of such well-known genetic disorders include Prader-Willi and Angelman syndromes, Williams syndrome, NF1 microdeletions, Sotos syndrome, Smith-Magenis syndrome, and the reciprocal deletions and duplications of Charcot Marie Tooth and HNPP (for reviews, see Emanuel and Shaikh 2001; Shaw and Lupski 2004).
There are a total of eight LCRs within 22q11. The four proximal LCRs have been extensively characterized, given their involvement in recurrent rearrangements of 22q11 that lead to DGS/VCFS (Edelmann et al. 1999
We hypothesized that deletion breakpoints falling within the smaller, less complex distal LCRs would be more amenable to characterization at the nucleotide level. This data could, in turn, help identify the sequences and mechanisms involved in 22q11 LCR-mediated rearrangements. Here, we examined two de novo deletions of 22q11 each flanked by at least one smaller, less complex LCR. One deletion is flanked by LCR-D and LCR-E and confers a phenotype with features of the DGS/VCFS spectrum (Saitta et al. 1999
The deletions are flanked by distal 22q11 LCRs For patient CH98-18, we initially identified a de novo deletion of the area adjacent to but not overlapping with the common DGS/VCFS region (Saitta et al. 1999 ). We further localized the deletion by FISH to an  1 Mb region between LCR-D and LCR-E (Fig. 1; Saitta et al. 1999), using a series of cosmids that were taken from a chromosome 22-specific cosmid library (LL22NCO3) (Fig. 1A). The results of one of these FISH experiments using probes c11e6 and cos82 are shown in Figure 1B. Patient CH03-29 had prior clinical testing with a normal karyotype, but subtelomeric FISH testing (Telvysion; Vysis) showed a deletion of the BCR probe that was used as an interstitial control signal for the 22q telomere. He also had normal FISH results for the DGS/VCFS region using both the N25 and TUPLE probes (Vysis). Further FISH studies suggested that the 650 kb deletion in CH03-29 was between LCR-E and LCR-F (Fig. 1A). The FISH experiment using probes c102d1 and cos82 is shown in Figure 1C.
Sequence analysis and characterization of LCR-E and LCR-F LCR-A through LCR-D in 22q11 have been previously implicated in mediating its deletions. The sequence-based analysis and structural configuration of LCR-D has been described previously (Fig. 2A; Shaikh et al. 2000 ). We performed a similar analysis of LCR-E and LCR-F to delineate their structure and organization. LCR-E is the smallest and least complex of the eight modular LCRs (A–H) in 22q11, essentially containing only two duplicated modules, one with the BCRL marker and the other with the GGTL marker described previously (Fig. 2B; Shaikh et al. 2000). The duplicated modules in LCR-E are intrachromosomal as the paralogs are found only on chromosome 22. The total size of duplicated sequence within LCR-E is 35 kb, corresponding to chromosomal coordinates chr22: 21,287,634–21,322,132 (NCBI build 35, May 2004) and localizes 1 Mb distal to the telomeric end of LCR-D.
LCR-F is larger and more complex, containing both inter- and intrachromosomal modules interspersed with small stretches of unique sequence. It extends over 370 kb, corresponding to chromosomal coordinates chr22: 21,973,629–22,345,857. Proximally, LCR-F begins within the 3'-end of the 135 kb BCR gene, whose 5'-regions contain the breakpoints of the Philadelphia chromosome translocations found in chronic myeloid leukemia and acute lymphocytic leukemia (Emanuel et al. 1984; Groffen et al. 1984). An 14 kb fragment consisting of the 3'-end of the BCR gene (BCRL) (Fig. 2C, blue box) is found within all of the other 22q11 LCRs, except for LCR-B, which contains a gap in its sequence. LCR-F also has a duplicated module containing the marker NF1L, with paralogs in LCRs A, B, and D (Shaikh et al. 2000; Fig. 2C, green box). There are duplicated modules within LCR-F with paralogs on chromosome 22, but not within the other 22q11 LCRs (Fig. 2C, modules M1–M4). In addition, LCR-F contains modules that are duplicated on chromosomes 7, 9, and 16 (Fig. 2C). Based on our FISH data, LCR-E appeared to be involved in the rearrangements of both CH98-18 and CH03-29. We took advantage of the simpler structure and smaller size of LCR-E to predict the likely substrates for nonallelic homologous recombination in patient CH98-18 between LCR-E and the highly complex LCR-D. Similarly, we compared the structures of LCR-E and the large, complex LCR-F to predict the likely breakpoint region for the deletion in patient CH03-29.
Cloning and molecular analysis of the deletion breakpoints We used a PCR-based strategy and somatic cell hybrids (S. Saitta, unpubl.) that contained either the deleted homolog (hybrid H1) or the normal homolog (hybrid H5) to determine if the proximal breakpoint of the deletion was within BCRL-D1 or in BCRL-D2 (see Supplemental Results). PCR amplimer DP1 (Fig. 2A) was amplified from both hybrids (Supplemental Fig. S1B). Amplimer BN1 (Fig. 2A) was deleted in hybrid H1 and present in hybrid H5 (Supplemental Fig. S1B), localizing the proximal deletion breakpoint to BCRL-D1.
Southern hybridization of digested genomic DNA from CH98-18 was performed with probe DP2, distal to LCR-E (Fig. 3A). DNA digested with NdeI yielded a novel
We next designed long-range PCR primers to span across the putative breakpoint junction (details in Supplemental Results). Primers BDfor and BErev were used to amplify genomic DNA (Fig. 4) from CH98-18, the H1 and H5 hybrids, and from cosmid 31f3 (spans LCR-E). Fragment 1 was present only in H1 and in total genomic DNA from CH98-18, while absent in hybrid H5, consistent with a putative junction fragment. Fragments 1 and 2 from H1 and fragment 3 from H5 (Fig. 4A) were gel-purified, cloned, and sequenced. The sequence of fragment 2 was analyzed using BLAST and was shown to match a distal region on 22q11 corresponding to LCR-G, displaying 99.5% sequence identity. The sequence of fragment 3 was shown to be identical to a region within LCR-E, derived from the patient’s normal, intact 22q homolog. BLAST analysis of fragment 1 instead confirmed its sequence as the junction fragment.
The total size of fragment 1 was 6083 bp of which the first 4354 bp shared the greatest sequence identity with BCRL-D1, while base pairs 4355–6083 shared the greatest sequence identity with nucleotides 43,880–45,609 of BAC clone b1000E4 (GenBank no. AC002308 [GenBank] ) that encompasses BCRL-E. We selected a 300 bp region (4101–4400) around the putative deletion breakpoints for further analysis, by performing multiple sequence alignments with CLUSTALW (http://www.ebi.ac.uk/clustalw; Higgins et al. 1996 ). A sequence variant specific to the BCRL-D1 paralog is present at position 4245 of the cloned junction fragment and BCRL-E specific sequence variants at positions 4355 and 4393 (Fig. 4B). Thus, the crossover most likely occurred in a 109 bp interval within BCRL-D1 and BCRL-E corresponding to the region between nucleotides 4246 and 4354. This putative crossover region was further assessed for recombination-promoting motifs (Table 1) using consensus sequences described previously (Badge et al. 2000; Abeysinghe et al. 2003; Visser et al. 2005). The sequence contains a putative DNA polymerase arrest site and two putative immunoglobulin heavy chain class switch repeats within the 109 bp nucleotide breakpoint region.
The sequence from 4355 to 6083 bp of the rearranged fragment did not match perfectly with BCRL-E of the reference human genome represented by clone c31f3 (GenBank no. D87002 [GenBank] ). An apparent genomic polymorphism exists in the sequence of BCRL-E as represented by the two clones c31f3 and b1000e4, respectively. This is an insertion/deletion polymorphism involving an Alu element that led to an extra 323 bp present in b1000e4 (43982–44304 bp of AC002308 [GenBank] ). The location of the polymorphic Alu would be in chr22: 21,317,317–21,317,318 of the reference human genome. Comparative analysis with the chimpanzee (Pan troglodytes) shows the presence of this 323 bp sequence in clone RP43-41g5 (GenBank no. AC099533 [GenBank] ), which maps to this region (T.H. Shaikh, unpubl.). Our findings suggest that the ancestral sequence may have contained the 323 bp sequence, which then apparently became deleted in humans. The deletion polymorphism is significant as described below, since its presence may destabilize the region by generating palindromes.
The breakpoint regions in BCRL-D1 and b1000e4 were further analyzed using RepeatMasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker), which demonstrated that the 323 bp polymorphism in b1000e4 was due to the presence of an Alu repeat belonging to the Sg subfamily (Fig. 5). This Alu Sg repeat was present in an inverted orientation with respect to its neighboring Alu Y repeat. Such a configuration of two highly homologous, full-length Alu repeats creates a quasi-palindrome in this region. Using Mfold (Zuker 2003
Patient CH03-29 Sequence alignment between LCR-E and LCR-F was performed using BLAST (Altschul et al. 1990 ), which demonstrated that BCRL-E shared 97% sequence identity with BCRL-F. These two modules are in the same direct orientation to each other, and are the only duplicated module shared between LCR-E and LCR-F. This became the best candidate region to mediate NAHR, resulting in the deletion found in CH03-29. There were no somatic cell hybrids available for this patient, thus we used long-range PCR to directly amplify the junction fragment in CH03-29 using total genomic DNA. We designed PCR primers to amplify a rearranged fragment potentially generated by NAHR between BCRL-E and BCRL-F. Primers BEfor1 and BFrev were designed to generate an 15 kb PCR product unique to patient CH03-29 representing the deletion junction fragment (primer details in Supplemental Results). In addition, we designed a nested primer, BEfor2, that would generate an 7 kb PCR product when used with primer BFrev representing the junction fragment.
Long-range PCR with BEfor1 and BFrev did not yield a visually detectable PCR fragment, presumably because of the large (15 kb) product size. Instead, BEfor2 and BFrev, when used as PCR primers, yielded the expected
LCRs of chromosome 22q11 have been implicated in the rearrangements associated with DGS/VCFS and CES (Edelmann et al. 1999 ; Shaikh et al. 2000; McDermid and Morrow 2002). The complexity and large size of the LCRs responsible for these disorders have prevented the precise localization of the deletion breakpoints within the proximal 22q11 LCRs. Our strategy to analyze deletions of 22q11 mediated by the distal, lower complexity LCRs enabled us to successfully identify breakpoints within 22q11 LCRs. The deletion breakpoints we analyzed are each located within a duplicated module containing the BCRL marker, suggesting that the sequences within this module may predispose the region to rearrangement. Interestingly, the region corresponding to the BCRL module within the 22q11 LCRs was previously predicted to be a potential rearrangement hotspot within LCR-A (LCR22-2) and LCR-D (LCR22-4) (Pavlicek et al. 2005). This interval, designated  BCR, is enriched in shared polymorphic sites (SPSs) and poor in paralogous sequence variants (PSVs), suggesting it as a region for gene conversion and consequently for meiotic crossover and rearrangement (Hurles et al. 2004; Pavlicek et al. 2005). This congruence between the predicted region and our reported breakpoint regions is highly suggestive.
The BCRL module is the only duplicated module common to almost all (7/8) 22q11 LCRs in the reference human genome sequence (Fig. 7). The relative orientation of the BCRL modules may further predict which LCRs will mediate NAHR, since it has been suggested that LCRs in a direct orientation mediate deletions and duplications (Shaffer and Lupski 2000
We narrowed down the putative crossover regions in CH98-18 and CH03-29 to 109 bp and 225 bp, respectively. The breakpoint sequences were tested further to determine if the genomic architecture or repetitive DNA content within each BCRL module predisposed the region to rearrangement. Alu-mediated recombination has been shown to cause deletions that lead to many human diseases (Deininger and Batzer 1999 ). We found no repetitive elements including Alus within the putative crossover regions in CH98-18 and CH03-29. However, while Alu repeats are not directly involved at the deletion breakpoints, analysis of the junction fragment in CH98-18 suggests that an apparent Alu-based insertion/deletion polymorphism may play a role in creating genomic instability. We identified the Alu polymorphism within the sequence of BCRL-E, based on sequences of clones obtained from different genomic libraries. The presence of such a polymorphism in close proximity to a deletion breakpoint is intriguing, but more importantly, the polymorphism leads to the juxtaposition of two highly homologous Alus in an inverted orientation with respect to each other. Inverted Alu elements are rare within the human genome (Stenger et al. 2001) but are known to be hotspots for genomic instability (Gebow et al. 2000; Lobachev et al. 2000; Stenger et al. 2001). This Alu polymorphism may represent a genomic variant that predisposes BCRL-E to genomic instability resulting in deletions.
Computational analysis with Mfold predicted the formation of cruciform structures resulting from the inverted Alus. Palindrome-mediated cruciforms have been shown to be involved in several constitutional translocation breakpoints (Kurahashi et al. 2000
The deletion breakpoint sequences were also tested for the presence of recombination promoting motifs that have been previously identified at or near deletion and translocation breakpoints (Badge et al. 2000
Our results provide confirmation that distal 22q11 LCRs (LCR-E–LCR-H) can and do mediate rearrangements leading to genomic disorders. More patients may exist who harbor microdeletions or microduplications in 22q11 mediated by the distal LCRs that are undetected by current techniques used in clinical testing. Recently, microarray-based CGH has been used to detect copy number alterations in 22q11 and across the entire genome (Mantripragada et al. 2004
Patient samples Clinical histories and family data were obtained from medical records. Each patient was evaluated by a clinical geneticist, and each had a normal karyotype and normal chromosome 22q11.2 FISH using commercial DiGeorge syndrome probes. Patient CH98-18 has been reported previously (Saitta et al. 1999 ) and had microcephaly, hypertelorism, cardiac defect, and bifid uvula. Patient CH03-29 displayed developmental delay, speech apraxia, short stature, hypertelorism, down-turned mouth, and pectus excavatum.
PCR
Southern blots
Long-range PCR
Patient CH 98-18
The amplification profile included 2 min of initial denaturation at 94°C, followed by 10 cycles of 94°C for 10 sec, 64°C for 30 sec, and 68°C for 7 min. A subsequent 18 cycles of the same segments was performed followed by a 7 min extension at 68°C. The PCR products were electrophoresed on a 1% agarose gel in 0.5x TBE using a pulsed-field apparatus (CHEF Mapper; Bio-Rad).
Patient CH 03-29
The amplification profile for the BEfor2 and BFrev primers included 2 min of initial denaturation at 94°C, followed by 10 cycles of 94°C for 10 sec, 66°C for 30 sec, and 68°C for 7 min. A subsequent 18 cycles of the same segments was performed followed by a 7 min extension at 68°C. The PCR products were electrophoresed on a 1% agarose gel in 1x TBE using a standard gel electrophoresis apparatus.
This work was supported by the NIH, K08 HL04487 (S.C.S.), the Physician Scientist award program, P30 HD28815 (S.C.S.), and by R01 GM64725 (T.H.S.).
5 These authors contributed equally to this work. 
E-mail sulagna{at}mail.med.upenn.edu; fax (215) 590-3764. [Supplemental material is available online at www.genome.org. The sequence data have been submitted to GenBank under accession nos. EF025176–EF025177.] Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5986507
Abeysinghe, S.S., Chuzhanova, N., Krawczak, M., Ball, E.V., and Cooper, D.N. 2003. Translocation and gross deletion breakpoints in human inherited disease and cancer I: Nucleotide composition and recombination-associated motifs. Hum. Mutat. 22: 229–244.[CrossRef][Medline] Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.[CrossRef][Medline] Bacolla, A., Jaworski, A., Larson, J.E., Jakupciak, J.P., Chuzhanova, N., Abeysinghe, S.S., O’Connell, C.D., Cooper, D.N., and Wells, R.D. 2004. Breakpoints of gross deletions coincide with non-B DNA conformations. Proc. Natl. Acad. Sci. 101: 14162–14167. Badge, R.M., Yardley, J., Jeffreys, A.J., and Armour, J.A. 2000. Crossover breakpoint mapping identifies a subtelomeric hotspot for male meiotic recombination. Hum. Mol. Genet. 9: 1239–1244. Bayes, M., Magano, L.F., Rivera, N., Flores, R., and Perez Jurado, L.A. 2003. Mutational mechanisms of Williams-Beuren syndrome deletions. Am. J. Hum. Genet. 73: 131–151.[CrossRef][Medline] Bi, W., Park, S.S., Shaw, C.J., Withers, M.A., Patel, P.I., and Lupski, J.R. 2003. Reciprocal crossovers and a positional preference for strand exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2. Am. J. Hum. Genet. 73: 1302–1315.[CrossRef][Medline] Burn, J. and Goodship, J. 1996. Developmental genetics of the heart. Curr. Opin. Genet. Dev. 6: 322–325.[CrossRef][Medline] Cao, X., Eu, K.W., Seow-Choen, F., Zhao, Y., and Cheah, P.Y. 2001. Topoisomerase-I- and Alu-mediated genomic deletions of the APC gene in familial adenomatous polyposis. Hum. Genet. 108: 436–442.[CrossRef][Medline] Deininger, P.L. and Batzer, M.A. 1999. Alu repeats and human disease. Mol. Genet. Metab. 67: 183–193.[CrossRef][Medline] Edelmann, L., Pandita, R.K., Spiteri, E., Funke, B., Goldberg, R., Palanisamy, N., Chaganti, R.S., Magenis, E., Shprintzen, R.J., and Morrow, B.E. 1999. A common molecular basis for rearrangement disorders on chromosome 22q11. Hum. Mol. Genet. 8: 1157–1167. Emanuel, B.S. and Shaikh, T.H. 2001. Segmental duplications: An ‘expanding’ role in genomic instability and disease. Nat. Rev. Genet. 2: 791–800.[Medline] Emanuel, B.S., Selden, J.R., Wang, E., Nowell, P.C., and Croce, C.M. 1984. In situ hybridization and translocation breakpoint mapping. I. Nonidentical 22q11 breakpoints for the t(9;22) of CML and the t(8;22) of Burkitt lymphoma. Cytogenet. Cell Genet. 38: 127–131.[Medline] Gebow, D., Miselis, N., and Liber, H.L. 2000. Homologous and nonhomologous recombination resulting in deletion: Effects of p53 status, microhomology, and repetitive DNA length and orientation. Mol. Cell. Biol. 20: 4028–4035. Gotter, A.L., Shaikh, T.H., Budarf, M.L., Rhodes, C.H., and Emanuel, B.S. 2004. A palindrome-mediated mechanism distinguishes translocations involving LCR-B of chromosome 22q11.2. Hum. Mol. Genet. 13: 103–115. Groffen, J., Stephenson, J.R., Heisterkamp, N., de Klein, A., Bartram, C.R., and Grosveld, G. 1984. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36: 93–99.[CrossRef][Medline] Higgins, D.G., Thompson, J.D., and Gibson, T.J. 1996. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266: 383–402.[Medline] Hurles, M.E., Willey, D., Matthews, L., and Hussain, S.S. 2004. Origins of chromosomal rearrangement hotspots in the human genome: Evidence from the AZFa deletion hotspots. Genome Biol. 5: R55.[CrossRef][Medline] Hyrien, O. 2000. Mechanisms and consequences of replication fork arrest. Biochimie 82: 5–17.[Medline] Koolen, D.A., Vissers, L.E., Pfundt, R., de Leeuw, N., Knight, S.J., Regan, R., Kooy, R.F., Reyniers, E., Romano, C., Fichera, M., et al. 2006. A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism. Nat. Genet. 38: 999–1001.[CrossRef][Medline] Kurahashi, H., Shaikh, T.H., Hu, P., Roe, B.A., Emanuel, B.S., and Budarf, M.L. 2000. Regions of genomic instability on 22q11 and 11q23 as the etiology for the recurrent constitutional t(11;22). Hum. Mol. Genet. 9: 1665–1670. Lobachev, K.S., Stenger, J.E., Kozyreva, O.G., Jurka, J., Gordenin, D.A., and Resnick, M.A. 2000. Inverted Alu repeats unstable in yeast are excluded from the human genome. EMBO J. 19: 3822–3830.[CrossRef][Medline] Lopez-Correa, C., Dorschner, M., Brems, H., Lazaro, C., Clementi, M., Upadhyaya, M., Dooijes, D., Moog, U., Kehrer-Sawatzki, H., Rutkowski, J.L., et al. 2001. Recombination hotspot in NF1 microdeletion patients. Hum. Mol. Genet. 10: 1387–1392. Mantripragada, K.K., Tapia-Paez, I., Blennow, E., Nilsson, P., Wedell, A., and Dumanski, J.P. 2004. DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets. Int. J. Mol. Med. 13: 273–279.[Medline] McDermid, H.E. and Morrow, B.E. 2002. Genomic disorders on 22q11. Am. J. Hum. Genet. 70: 1077–1088.[CrossRef][Medline] McTaggart, K.E., Budarf, M.L., Driscoll, D.A., Emanuel, B.S., Ferreira, P., and McDermid, H.E. 1998. Cat eye syndrome chromosome breakpoint clustering: Identification of two intervals also associated with 22q11 deletion syndrome breakpoints. Cytogenet. Cell Genet. 81: 222–228.[CrossRef][Medline] Michel, B. 2000. Replication fork arrest and DNA recombination. Trends Biochem. Sci. 25: 173–178.[CrossRef][Medline] Ming, J.E., Geiger, E., James, A.C., Ciprero, K.L., Nimmakayalu, M., Zhang, Y., Huang, A., Vaddi, M., Rappaport, E., Zackai, E.H., et al. 2006. Rapid detection of sub-microscopic chromosomal rearrangements in children with multiple congenital anomalies using high density oligonucleotide arrays. Hum. Mutat. 27: 467–473.[CrossRef][Medline] Pavlicek, A., House, R., Gentles, A.J., Jurka, J., and Morrow, B.E. 2005. Traffic of genetic information between segmental duplications flanking the typical 22q11.2 deletion in velo-cardio-facial syndrome/DiGeorge syndrome. Genome Res. 15: 1487–1495. Rauch, A., Pfeiffer, R.A., Leipold, G., Singer, H., Tigges, M., and Hofbeck, M. 1999. A novel 22q11.2 microdeletion in DiGeorge syndrome. Am. J. Hum. Genet. 64: 659–666.[CrossRef][Medline] Ravnan, J.B., Tepperberg, J.H., Papenhausen, P., Lamb, A.N., Hedrick, J., Eash, D., Ledbetter, D.H., and Martin, C.L. 2006. Subtelomere FISH analysis of 11,688 cases: An evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities. J. Med. Genet. 43: 478–489. Reiter, L.T., Hastings, P.J., Nelis, E., De Jonghe, P., Van Broeckhoven, C., and Lupski, J.R. 1998. Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients. Am. J. Hum. Genet. 62: 1023–1033.[CrossRef][Medline] Saitta, S.C., McGrath, J.M., Mensch, H., Shaikh, T.H., Zackai, E.H., and Emanuel, B.S. 1999. A 22q11.2 deletion that excludes UFD1L and CDC45L in a patient with conotruncal and craniofacial defects. Am. J. Hum. Genet. 65: 562–566.[CrossRef][Medline] Saitta, S.C., Harris, S.E., Gaeth, A.P., Driscoll, D.A., McDonald-McGinn, D.M., Maisenbacher, M.K., Yersak, J.M., Chakraborty, P.K., Hacker, A.M., Zackai, E.H., et al. 2004. Aberrant interchromosomal exchanges are the predominant cause of the 22q11.2 deletion. Hum. Mol. Genet. 13: 417–428. Sambrook, J. and Russell, D.W. 2001. Molecular cloning: A laboratory manual,, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shaffer, L.G. and Lupski, J.R. 2000. Molecular mechanisms for constitutional chromosomal rearrangements in humans. Annu. Rev. Genet. 34: 297–329.[CrossRef][Medline] Shaikh, T.H., Kurahashi, H., Saitta, S.C., O’Hare, A.M., Hu, P., Roe, B.A., Driscoll, D.A., McDonald-McGinn, D.M., Zackai, E.H., Budarf, M.L., et al. 2000. Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: Genomic organization and deletion endpoint analysis. Hum. Mol. Genet. 9: 489–501. Shaikh, T.H., Kurahashi, H., and Emanuel, B.S. 2001. Evolutionarily conserved low copy repeats (LCRs) in 22q11 mediate deletions, duplications, translocations, and genomic instability: An update and literature review. Genet. Med. 3: 6–13.[Medline] Sharp, A.J., Hansen, S., Selzer, R.R., Cheng, Z., Regan, R., Hurst, J.A., Stewart, H., Price, S.M., Blair, E., Hennekam, R.C., et al. 2006. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat. Genet. 38: 1038–1042.[CrossRef][Medline] Shaw, C.J. and Lupski, J.R. 2004. Implications of human genome architecture for rearrangement-based disorders: The genomic basis of disease. Hum. Mol. Genet. 13: R57–R64. Shaw, C.J., Withers, M.A., and Lupski, J.R. 2004. Uncommon deletions of the Smith-Magenis syndrome region can be recurrent when alternate low-copy repeats act as homologous recombination substrates. Am. J. Hum. Genet. 75: 75–81.[CrossRef][Medline] Shaw-Smith, C., Pittman, A.M., Willatt, L., Martin, H., Rickman, L., Gribble, S., Curley, R., Cumming, S., Dunn, C., Kalaitzopoulos, D., et al. 2006. Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability. Nat. Genet. 38: 1032–1037.[CrossRef][Medline] Stary, A. and Sarasin, A. 1992. Molecular analysis of DNA junctions produced by illegitimate recombination in human cells. Nucleic Acids Res. 20: 4269–4274. Stenger, J.E., Lobachev, K.S., Gordenin, D., Darden, T.A., Jurka, J., and Resnick, M.A. 2001. Biased distribution of inverted and direct Alus in the human genome: Implications for insertion, exclusion, and genome stability. Genome Res. 11: 12–27. Urban, A.E., Korbel, J.O., Selzer, R., Richmond, T., Hacker, A., Popescu, G.V., Cubells, J.F., Green, R., Emanuel, B.S., Gerstein, M.B., et al. 2006. High-resolution mapping of DNA copy alterations in human chromosome 22 using high-density tiling oligonucleotide arrays. Proc. Natl. Acad. Sci. 103: 4534–4539. Visser, R., Shimokawa, O., Harada, N., Kinoshita, A., Ohta, T., Niikawa, N., and Matsumoto, N. 2005. Identification of a 3.0-kb major recombination hotspot in patients with Sotos syndrome who carry a common 1.9-Mb microdeletion. Am. J. Hum. Genet. 76: 52–67.[CrossRef][Medline] Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31: 3406–3415.
Received September 22, 2006; accepted in revised format January 22, 2007.  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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Abstract
Results
G values (Supplemental Fig. S2). The palindromic Alus are in close proximity to the putative crossover point: only 168 bp away in BCRL-D1 and 182 bp away in b1000e4. 
frameshift hotspot, one putative topoisomerase I consensus sequence, and one potential DNA polymerase 
frameshift hotspot within the 225 nucleotide breakpoint region. 
