The Genom of Homo sapiens.pdf

44 RIETHMAN ET AL.gions were unavailable until now and because currentmethods for detecting and analyzing large-scale variationare limited. FISH-based detection of polymorphism islimited to DNA contained in the cloned probe and provideslittle information with respect to the real size of thepolymorphic segment. Nonetheless, it has provided informationon subtelomeric regions that are clearly variable(Ijdo et al. 1992; Trask et al. 1998). Conventional PFGEanalysis of DNA fragments enabled the initial observationsof large-scale variation at the 16p telomere (Wilkieet al. 1991), but this method is limited by the variablemethylation of genomic DNA and the sensitivity of infrequentlycutting restriction enzymes to CpG methylation.We have used a site-specific DNA cleavage method(RecA-assisted restriction endonuclease [RARE] cleavage;Riethman et al. 1997) combined with pulsed-field gelelectrophoresis (PFGE) analysis of the cleaved large DNAfragments to analyze subtelomere structure. A RAREcleavage experiment targeting a single genomic site in asubtelomeric region is expected to release a telomere-terminalfragment of genomic DNA; the size of this fragmentcorresponds to the distance from the cleavage site to theend of the chromosome. This simple principle makesRARE cleavage mapping an ideal method for physicallymapping telomere regions, simplifies validation of half-YAC clone structure, and enables the systematic analysisof large polymorphisms in human subtelomeric regions.Table 1 summarizes the current state of the experimentalcharacterization of human subtelomeric variation. Onthe basis of current data and the number of telomeres forwhich no large-scale variation data are yet available,~2–4 large-scale length variants are expected to exist ateach of 20–25 human telomeres. This is, however, a verypreliminary and rough estimate, and a proper assessmentof the occurrence and frequency of large subtelomericvariants awaits a more systematic analysis.Figure 2 shows models of large-scale variation leadingto large-length polymorphisms at human telomeres. Themodels are based on published mapping studies (see Table1) as well as our unpublished results. In Figure 2a, the allelesvary by addition of a Srpt segment to an existingtelomere. Some of the 11p, 16p, and 8p variants appear tohave this structure. In Figure 2b, the alleles vary in bothSrpt content and Srpt organization, without a simple relationshipto each other in the variant region. Some of the 6pand 8p variants appear to follow this model, as does a19pvariant. In Figure 2c, the two alleles diverge just before theSrpt region begins, leading to a large insertion/deletionpolymorphism in distal 1-copy DNA. Evidence for thissort of variation exists at the 2q telomere. In Figure 2d, thetwo alleles are similar except for an insertion/deletionpolymorphism contained entirely within the segmentallyduplicated DNA. Evidence for this exists near the 14qtelomere where duplicated IgG VH genes are located. InFigure 2e, the alleles vary by insertion/deletion polymorphismof tandemly repeated DNA tracts located within theSrpt region. Evidence for this model comes from mappingthe D4Z4 repeat tract near the 4q and 10q telomeres. Combinationsof these five classes of alternative subtelomericsequence organization are possible, as are additional typesnot yet detected.HUMAN (TTAGGG)n-ADJACENT DNAThe critical DNA regions required for developingreagents for single-telomere (TTAGGG)n tract-length assaysare those immediately adjacent to the terminal(TTAGGG)n tract (subterminal sequences). Within thepast 6 months there has been a dramatic expansion in theavailability of these sequences as work in our own lab andelsewhere has resulted in completion of telomere regionsfor many reference alleles (see Table 1). Sequences formultiple variant subtelomere regions will continue to beadded to the databases in the near future as our own andother telomere mapping and sequencing projectsprogress, providing the raw material for PCR-based singletelomere length assays (Forstemann et al. 2000; Bairdet al. 2003). In these assays, a sequence for priming PCRtoward the centromere from the natural (TTAGGG)ntract terminus is added (either by ligation or by a terminaltransferase-based method), and a second primer derivedfrom subterminal DNA and oriented toward the telomereis paired with the first primer for PCR. A hybridizationprobe derived from subterminal DNA distal to the telomere-orientedprimer is used to detect the PCR product.The specificity of the PCR for a single telomere (or a specificallele of a telomere) relies on the selection of thetelomere-oriented primer from what are often closely relatedsubterminal sequences; thus, as a database of subtelomericand subterminal sequences is expanded, thepower to select specific primers for PCR-based telomerelengthassays will increase.Figure 3 illustrates the three most common types ofsubterminal sequence organizations found in fully sequencedtelomere alleles so far. The sequence organizationshown in Figure 3a is the simplest and most straightforwardfor PCR-based assay design. There is a short(2–10 kb) region of low-copy Srpt sequence immediatelyadjacent to the (TTAGGG)n tract, followed by 1-copyDNA from which unique probes and primers can be derived.The original STELA assay was derived from sucha telomere (Xp/Yp telomere; Baird et al. 2003), and allele-specificSTELA and telomere-PCR (Forstemann etal. 2000) assays can be prepared by using a telomere-orientedprimer that is allele-specific (A_sp in Fig. 3a).Telomeres in this class include 7qtel, 8qtel, 11qtel,14qtel, 18qtel, and Xp/Yptel, each of which already has afully sequenced or nearly sequenced reference allele.Figure 3b shows the TelBam11 class of subterminallow-copy repeat sequence (Brown et al. 1990) that isfound on 10–20 telomeres in each individual, and is representedon fully sequenced reference alleles for 10qteland 21qtel. In this case, a DNA hybridization probe weisolated (HC1208) that is specific for this low-copy Srptsequence recognizes all members of this subtelomeric repeatfamily; the specificity of the telomere-length assayfor single telomeres and for single alleles must thereforecome entirely from the telomere-oriented primer (A_sp),which must therefore be carefully selected from divergentsequences within the adjacent subtelomeric DNA. Preliminaryresults indicate that TelBam11-associated sequencestypically display 90–98% nucleotide sequence identity,permitting sufficient divergence for development of