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CHR refers to the chromosome for the insertion.

2X and 3X refer to genes that have had two or three insertions, respectively.

CHR refers to the chromosome for the insertion.

2X and 3X refer to genes that have had two or three insertions, respectively.

lead to mistakes in DNA repair and other DNA damage. Such events would be the result of an attempted Alu insertion, but would leave no evidence of the role of the Alu element. Thus, at this point we believe that we underestimate the role of mobile elements in recombining and damaging the human genome.

POSTINSERTION DAMAGE Recombination

Alu elements may continue to contribute to genetic instability even if they do not initially damage a gene. A common form of secondary damage is owing to unequal homologous recombination, but several other types of mutations can alter the properties of Alu elements to create damage.

Alu elements may contribute to recombination events in several ways. The best understood is through either a deletion using the single-strand annealing reaction (Fig. 2D) or a reciprocal, unequal homologous recombination (Fig. 4A), which can cause either duplications or deletions of the segments between the Alu elements that recombine. The recombination event does not have to be reciprocal. For instance, if cells use the single-strand annealing pathway of recombination, nearby homologous Alu elements may recombine causing only deletions. These types of homologous recombination events have been estimated to cause at least 0.3% of human genetic disease (2). However, as the majority of larger genomic rearrangements have not been characterized to this level, it seems likely that this represents an underestimate.

There are several studies suggesting that Alu elements located near one another in an inverted orientation may be even more destabilizing than those in a direct orientation (17-19). How-

Fig. 4. Unequal Alu-Alu recombination. (A) WhenAlu elements are located in the same orientation near one another in the genome, it is possible for an unequal homologous recombination event to occur that can give rise to either a duplication or deletion of the sequences between the Alus. A hybrid Alu element is usually formed at the point of recombination. (B) When Alu elements are in the inverted orientation, they also seem to trigger recombination in their vicinity, but the recombination junctions do not seem to be driven by recombination so that the resulting products can be variable.

Fig. 4. Unequal Alu-Alu recombination. (A) WhenAlu elements are located in the same orientation near one another in the genome, it is possible for an unequal homologous recombination event to occur that can give rise to either a duplication or deletion of the sequences between the Alus. A hybrid Alu element is usually formed at the point of recombination. (B) When Alu elements are in the inverted orientation, they also seem to trigger recombination in their vicinity, but the recombination junctions do not seem to be driven by recombination so that the resulting products can be variable.

ever, the instability caused by the inverted Alu elements (Fig. 4B) does not create a predictable junction that allows individual recombination events to be definitively defined as being caused by Alu elements. Recombination can occur at various locations around both Alu elements. Thus, it is difficult to distinguish between Alu-induced recombination events of this type, and Alu-unrelated nonhomologous end-joining events. However, it has been suggested that secondary structures may contribute to the majority of recombination junctions (20), and Alu elements represent one of the most abundant elements that could consistently contribute to such secondary structures.

We have previously reported a broad range of genes that have undergone homologous recombination events leading to genetic defects (2). These represent a broad variety of genes, as Alu elements are spread throughout essentially all genes. However, there are a few genes with unusually high levels of Alu-Alu recombination events. This includes a large number of events in the LDLR and C1 inhibitor loci. However, there are also seven different recombination events leading to breast cancer in the BRCA1 gene (21-25), three mutations in the MSH2 gene that may represent as much as 10% of the defects in that gene (26,27), and a duplication and a deletion in MSH6 (28). The majority of cases of acute myelogenous leukemia that do not involve a visible translocation have been shown to involve Alu-Alu-mediated duplication events in the MLL gene (29). Although inter-chromosomal translocations generally do not

Fig. 5. Map of the Alu-Alu recombination junctions found in an assortment of recombination events. The darker dashed lines represent those occurring in the LDLR gene. The lighter dashed lines represent those occurring in the globin genes. The solid gray lines represent those that occurred in theMLL gene leading to acute myelogenous leukemia and the solid dark lines represent recombination events in an assortment of other genes. The length of the lines represents the uncertainty in the exact point of the recombination events relative to the schematic map of theAlu dimer shown. The arrows in the dimer portions represent the A-rich regions.

involve Alu-Alu-mediated translocations, there is now some evidence that many of these cases have additional, smaller rearrangements in the MLL gene that may be Alu-mediated (30). Recently, it has been suggested that up to 30% of mutations in the Fanconi anemia gene (FANCA) may be caused by Alu-Alu recombination events and that these types of events are not typically detected using the polymerase chain reaction strategies commonly in use (31). Thus, there is likely a strong ascertainment bias against detecting Alu-mediated genomic rearrangements.

Most of the Alu-Alu recombination events are between nearby elements, generally spanning distances of less than 50 kb and often only a few kb in length. There are also a number of cases of chromosomal translocations that suggest some involvement of Alu elements (20,32), but very few that involve Alu-Alu homologous recombination.

It has been proposed that Alu elements may contain specific sequences that trigger recombination (33). An analogy has been made to "chi-like" sites that may cause targeting of the recombination events within the upstream portion of the Alu element. The original data that triggered that hypothesis were mostly from recombination events in the LDLR gene that appear to cluster (Fig. 5). However, when a broader range of Alu-Alu recombination events are mapped, they occur dispersed throughout the Alu element, with only a modest predisposition to the left end. This includes both germline recombination events, as well as somatic events like those that contribute to AML (29). The apparent predisposition for the upstream end of Alu may be caused by numerous factors. One is that none of the positions that define the Alu subfamilies occur toward the upstream end of Alu and therefore Alu elements from different subfamilies may show fewer mismatches with one another at that end. Also, that region includes the RNA pol III promoter for the element and may have a more open structure, particularly with specific Alu elements that may be in the appropriate chromatin environment to favor expression (11).

Finally, whatever sequence features favored the insertion of the Alu at that location in the first place may lead to higher recombination rates near the end of the element.

Alternative Mutagenic Changes

In addition to a major contribution to genetic instability through insertion and recombination, Alu elements may also contribute to other forms of DNA damage. There are a growing number of reports that Alu elements may influence splicing of genes. Alu elements have inserted in a number of loci without causing any apparent defect, but point mutations in the Alu elements have led to activation of cryptic splice sites causing genetic defects (34,35). In addition, there is growing evidence that even Alu elements that are tolerated in genes may be contributing to a significant level of alternative and aberrant splicing (36-38). This leads to the inclusion of parts of Alu elements in a proportion of transcripts coming from the gene. In most cases these would be expected to result in defective RNAs and would, therefore, decrease the overall expression from those genes.

Although the A-tails of new Alu insertions appear to generally be homogeneous, they have been shown to commonly lead to the creation of more complex microsatellites over time (39). Although such changes would lead to frequent genetic polymorphism, the vast majority would be expected to be relatively harmless. One important example of a disease-causing change occurred in the middle A-rich region of an Alu in the frataxin gene (40). In one particular human, this region mutated into a GAA microsatellite. This GAA microsatellite created the permutation allele that grew through triplet repeat instability to a size where it somehow blocked transcription of the frataxin gene (41-43), leading to Friedreich ataxia in those carrying this microsatellite.

Other changes within these A-rich regions commonly generate the potential polyadenylation signal, AATAAA (44). This could lead to truncation of transcripts and disruption of gene expression.

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