Alu sequences represent, with about one million copies, the most abundant retrotransposable elements (RE) in the human genome. While observations in disease cases and experiments on ingegnerized model systems suggest a role of RE-associated mutations in human diseases and somatic mosaicism, the direct analysis of genetic variability associated with endogenous human Alu elements is difficult due to the lacking of a specific methodology for detecting new (sample-specific) elements over a background of one million similar copies. Here we tested two different strategies designed to perform an Alu insertion site profiling (AIP),that is to detect, on a whole-genome scale, loci associated with new Alu insertions.
Two AIP strategies have been tested, both based on the use of Affymetrix tiling microarrays and designed to preferentially target genomic sites associated to Alu Ya5, one of the youngest (mutationprone) Alu subfamilies. The two strategies differ in the method used to obtain the probes (labeled Alu-flanking DNA fragments) for microarray hybridization. Their efficacy has been tested by comparing genomic DNA samples from two healthy donors, using the 'Affymetrix Tiling Array 2.0R A' for chromosomes 1 and 6. Affymetrix Tiling Analysis Software (TAS) has been used for the analysis and sample-to-sample comparison.
The analysis of microarray data detected a series of loci likely associated to sample-specific Alu insertions on chromosomes 1 and 6. Part of the signals were detected by both AIP methods, others with only one of the two methods. As a confirmation of the specificity of signals, more than 15% of them coincides with previously known polymorphic Alu insertions. The remaining loci are candidate for carrying new, previously undetected Alu-insertion polymorphisms or mutations.
The tested AIP strategies represent promising methods to analyze Alu-linked genetic variability in different biological contexts (interindividual variability, somatic mosaicism, mutations in tumoral vs healthy cells). The experimental confirmation of the obtained results, based on the PCR amplification and sequencing of specific loci, is currently ongoing.