Lcrmediated i17q Formation

Isochromosome 17q [i(17q)] constitutes the most common isochromosome in human neo-plasia and has been described both as a primary and as a secondary abnormality, suggesting an important pathogenetic role in tumor development as well as in progression (32). The i(17q) is frequent in hematologic malignancies, in particular as a secondary abnormality in addition to the characteristic t(9;22)(q34;q11) in CML, but is also the most common chromosomal aberration in primitive neuroectodermal tumors and medulloblastomas (33).

Fioretos et al. (33) characterized the i(17q) in more detail, using fluorescence in situ hybridization (FISH) with several yeast artificial chomosome (YAC) clones on a series of leukemias, and showed that the i(17q), in most cases, was a dicentric isochromosome (isodicentric), and

Fig. 1. Schematic representation of the Isochromosome 17q [i(17q)] breakpoint region. At the top, an ideogram of chromosome 17 is depicted. In the middle, the localization of the breakpoint region with respect to the Smith-Magenis syndrome-REPs is shown. Below, the complex genome architecture of the breakpoint is shown with yellow and gray arrows referring to the REPA and REPB copies, respectively. The REPA and REPB copies are arranged according to their suggested orientation and structure. REPB2 is truncated when compared to REPB1 and REPB3 copies. Both REPA copies contain exons 1-3 of the GRAP gene, the remaining exons map telomeric to the REPA1 (not shown). This indicates that REPA2 originated from REPA1, the position of the putative evolutionary breakpoint is depicted with a vertical black arrow. Three U3b and two U3a genes map to the arrowhead portion of REPB and REPA, respectively. The blue arrowheads of REPAs and REPBs represent a nearly identical approx 4-kb sequence shared among both REPAs and REPBs. (Reproduced from ref. 8 with permission.)

Fig. 1. Schematic representation of the Isochromosome 17q [i(17q)] breakpoint region. At the top, an ideogram of chromosome 17 is depicted. In the middle, the localization of the breakpoint region with respect to the Smith-Magenis syndrome-REPs is shown. Below, the complex genome architecture of the breakpoint is shown with yellow and gray arrows referring to the REPA and REPB copies, respectively. The REPA and REPB copies are arranged according to their suggested orientation and structure. REPB2 is truncated when compared to REPB1 and REPB3 copies. Both REPA copies contain exons 1-3 of the GRAP gene, the remaining exons map telomeric to the REPA1 (not shown). This indicates that REPA2 originated from REPA1, the position of the putative evolutionary breakpoint is depicted with a vertical black arrow. Three U3b and two U3a genes map to the arrowhead portion of REPB and REPA, respectively. The blue arrowheads of REPAs and REPBs represent a nearly identical approx 4-kb sequence shared among both REPAs and REPBs. (Reproduced from ref. 8 with permission.)

not monocentric as suggested by conventional G-banding (34). Consistent with this observation, the breakpoints were shown to cluster within 17p11 implying that the i(17q) formally should be designated idic(17)(p11). Because of its less cumbersome and more descriptive designation, and also for historical reasons, "i(17q)" prevails in the literature. Interestingly, the majority of the 17p11 breakpoints were shown to occur within a 900 kb YAC clone (828b9) located in the Smith-Magenis syndrome (SMS) common deletion region (34,35), suggesting that this germ line genetically unstable region also could be of importance in generating the i(17q) somatic event.

Given the genetically unstable nature of the SMS common deletion region, further efforts were undertaken to delineate the i(17q) breakpoint region in 17p11. FISH mapping using a large set of bacterial artificial chromosome (BAC), P1 artificial chromosome, and fosmid clones covering the breakpoint, together with extensive sequence comparison and the analysis of restriction maps of individual clones, enabled the establishment of a physical map of an approx 240-kb genomic interval including the i(17q) breakpoint cluster region (Fig. 1). This region, localized between the middle and proximal SMS-REPs (Fig. 1), was shown to contain several interesting features. First, two types of distinct LCRs, designated REPA and REPB, present in two and three copies, respectively, were identified within this region. Secondly, their location and orientation with respect to each other strongly suggested that they play an important role in the formation of the i(17q) rearrangement. As outlined in Fig. 1, the two copies of REPA (REPA1 and REPA2) are both approx 38 kb in size, display 99.8% sequence identity, are arrayed inversely with respect to each other, and are separated by another LCR, the approx 48 kb REPB3. The organization of the three REPB copies (REPB1, -2, and -3) is more complex. REPB1 (approx 49 kb) and the truncated REPB2 (approx 43 kb) share 99.8% sequence identity and are inverted with respect to one another (inverted repeats [IRs]). They are separated by an approx 400-bp spacer region and, hence, represent a palindrome or cruciform structure. REPB3 (approx 48 kb) is oriented inversely with respect to REPB2 (99.8% identity). Another interesting feature is the presence of two U3b genes (U3b2 and U3b1) that are located in an inverted orientation in the center of this palindromic structure. A similar DNA structure of 8731 bp is present in the junction region between REPA1 and REPB3. The U3a or U3b genes are located at the head-portion of each REPA and REPB. They display more than 98.5% identity and belong to the U3 (RNU3) evolutionary conserved gene family of small nuclear RNAs required for the processing of pre-18S rRNA (36,37). The very high degree of similarities among the three REPBs and independently among two REPAs indicates that the segmental duplication events that generated these LCRs are of recent origin, as suggested by evolutionary studies of other highly homologous LCRs (38), or alternatively, undergo frequent gene conversion. A final remarkable feature of this region is the high content (approx 30%) of Alu repeats. Greater than 80% of the 4 kb flanking the center of the cruciform are repetitive sequences, with 50% consisting of Alu sequences. Furthermore, each of the five REPs is flanked by Alu sequences. Thus, as recently suggested (39), the high Alu repeat content could be important in the genesis of the large segmental duplications present in this region.

The large approx 86-kb long cruciform generated by REPB1 and REPB2 is a genomic architectural feature that potentially could trigger a recombination event resulting in the i(17q)-formation. Indeed, IRs or cruciforms are known to generate hairpins, facilitated by intrastrand pairing of complementary single-strand DNA sequences assembled in the lagging strand during DNA replication (40,41). A double-strand break (DSB) introduced in such a hairpin can result in deletions, or either inter- or intrachromosomal recombination events, mediated by the homologous recombination machinery (42). Critical factors implicated in the rate of such rearrangements are the extent of sequence identity between the inverted repeats, their size, and the length of the spacer (sequences present between the cruciform sequences). The rate of genomic rearrangements rises as the length of the repeat increases and the spacer sequence length decreases (43). The cruciform identified in the i(17q) breakpoint cluster region is characterized by two inverted repeats, approx 48 kb and approx 43 kb in length, separated by a 400-bp spacer. If broken, DNA ends generated in the hairpin/cruciform structures of the i(17q) breakpoint can trigger the DSB-repair (DSBR) machinery. A subsequent nonallelic homologous recombination (NAHR) event between the repeats with opposite orientations located in the two sister chromatids (i.e., sister chromatid exchange) can result in the formation of an isodicentric chromosome 17 and of an acentric fragment (Fig. 2) (8).

Similar to i(17q), breakpoint analyses of constitutional isochromosomes of the long arm of the X chromosome, "i(Xq)," and of the most common constitutional isodicentric chromosomes, i.e., "inv dup(15)" and "inv dup(22)," suggest that these isodicentric abnormalities also may result from NAHR between inverted LCRs (7). As to the role of IRs or cruciform in constitutional genomic rearrangements, such genome architecture features, albeit of smaller size, have been identified at the breakpoints of the most common non-Robertsonian translocation, t(11;22)(q23;q11) (44,45) and of the translocation t(17;22)(q11;q11) described in two

Fig. 2. Molecular mechanism for i(17q)-formation. (A) The division of a metaphase chromosome is shown. The double strand DNA of each chromatid is depicted in red and blue and the orientation with 3' and 5'. (B) The formation of a REPB1 and REPB2 palindrome with (C) subsequent breakage and (D) reunion between palindromes on sister chromatids results in the origin of both dicentric and acentric structures. (E) The dicentric and acentric structures after replication. The acentric material is lost in dividing cells because of the lack of a centromere. The (iso-) dicentric structure (isochromosome) is retained and will not become disrupted during anaphase because one of the two centromeres are inactivated as a result of their close proximity. Yellow and gray arrows refer to REPA and REPB copies, respectively, whereas the X depicts interchromatid mispairing of direct repeats. According to this model, i(17q) should formally be designated idic(17)(p11.2). (Reproduced from ref. 8 with permission.)

Fig. 2. Molecular mechanism for i(17q)-formation. (A) The division of a metaphase chromosome is shown. The double strand DNA of each chromatid is depicted in red and blue and the orientation with 3' and 5'. (B) The formation of a REPB1 and REPB2 palindrome with (C) subsequent breakage and (D) reunion between palindromes on sister chromatids results in the origin of both dicentric and acentric structures. (E) The dicentric and acentric structures after replication. The acentric material is lost in dividing cells because of the lack of a centromere. The (iso-) dicentric structure (isochromosome) is retained and will not become disrupted during anaphase because one of the two centromeres are inactivated as a result of their close proximity. Yellow and gray arrows refer to REPA and REPB copies, respectively, whereas the X depicts interchromatid mispairing of direct repeats. According to this model, i(17q) should formally be designated idic(17)(p11.2). (Reproduced from ref. 8 with permission.)

patients with neurofibromatosis type 1 (46,47). Moreover, genomic characterization of the AZFc region on chromosome Y, which is deleted in close to 20% of patients with oligo- or azoospermia, revealed a 3-Mb sequence that is deleted via homologous recombination events mediated by two direct repeats, present at the edges of this structure (48).

The breakpoint region of i(17q) resides within the SMS commonly deleted region and is flanked by the middle and proximal SMS-REPs. Furthermore, several other repeat elements have been reported telomeric and centromeric to the i(17q) breakpoint region (49-51). It is unclear why the breakpoints in i(17q) are clustered in the delineated 240-kb region and not in the other closely located repeats, but it could well be that only the complex nature of the breakpoint region allows this chromosomal abnormality to be formed, or, alternatively, that only such rearrangements provide a selective advantage to the neoplastic cells. The i(17q) breakpoint region could, of course, also facilitate erroneous meiotic recombination, but the resulting gross genomic imbalances (i.e., whole arm deletion of 17p and duplications of 17q), are unlikely to be compatible with normal embryonic development.

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