The Mosaic Structure of Human Pericentromeric DNA: A Strategy for Characterizing Complex Regions of the Human Genome

The human genome contains several different classes of repetitive elements that are categorized based largely on their copy number and their mode of propagation (Gardiner 1996; Vogt 1990). Two broad classes of repeats are generally recognized: interspersed and tandem repeat elements (Brown 1999). Tandemly repeated DNA, such as centromeric α-satellite and microsatellite DNA, is believed to expand and contract by mechanisms involving unequal crossing-over or replication slippage. In contrast, interspersed repetitive elements such as LINEs and SINEs, which comprise more than one-third of the total genome (Smit and Riggs 1996), are propagated via mechanisms of retrotransposition. Both classes of repeats are easily recognized as repetitive because of both their high copy number and their defined sequence characteristics. As more of the human genome is sequenced, it is becoming apparent that yet another class of repetitive DNA exists. Low copy repeat sequences are being discovered as many unique regions of the genome are found to have duplicate counterparts. Portions of some genes and even entire gene segments have been duplicated and exist at multiple, discrete locations within the genome (Eichler et al. 1996, 1997; van Deutekom et al. 1996; Regnier et al. 1997; Zimonjic et al. 1997; Trask et al. 1998; Horvath et al. 2000). Mapping and sequencing of the human genome indicates that a large number of these duplicated segments lie within pericentromeric and subtelomeric regions (Eichler 1998). These duplicated sequences, or paralogs, are nonprocessed. This suggests an underlying DNA transposition mechanism for their duplication and dispersal. Partial or complete paralogous genomic segments have been identified for several gene loci including ALD, SLC6A8, NF1, HERC2, KGF, FRG1, olfactory receptor, immunoglobulin variable κ-chain and immunoglobulin variable heavy chain segments. A large number of pericentromeric and subtelomeric regions (1p12, 1q12, 2p11, 3p13, 3qter, 15qter, 19pter, 9p11, 10p11, 13q11, 14q11, 15q11, 16p11, 17p11, 18p11, 18q11, 20p, 20q, 21p11, 21q11, 22p11, and 22q11) have been shown to be sites of these recent duplications (Zachau 1993; Arnold et al. 1996; Eichler et al. 1996, 1997; van Deutekom et al. 1996; Regnier et al. 1997; Reiter et al. 1997; Zimonjic et al. 1997; Potier et al. 1998; Ritchie et al. 1998; Trask et al. 1998; Amos-Landgraf et al. 1999; Brand-Arpon et al. 1999; Christian et al. 1999). These data suggest that the process of pericentromeric/subtelomeric duplication may be a general property of the human genome. The reason for this apparent location bias is unclear, although we have suggested that GC-rich repeat elements may play a role in the accumulation of duplicated segments within pericentromeric regions (Eichler et al. 1999). Paralogous regions can be very large (>150 kb) and can exhibit a high degree of sequence similarity (>98%) (Horvath et al. 2000; Orti et al. 1998). These properties make such segments particularly problematic for both mapping and sequencing of the human genome (Eichler 1998). Where the degree of similarity approaches levels observed for allelic variation, it is anticipated that it will be difficult to disentangle assembled sequence contigs constructed using whole shotgun sequencing approaches (Eichler 1998; Green 1997). Even high-throughput sequencing centers using traditional STS mapping and sequencing strategies have encountered difficulties building physical maps across such duplicated regions. Since large insert clones are linked into contiguous sets of overlapping clones based on short STS PCR products, the absence of unique STSs and/or the presence of a large number of clones from paralogous loci within a single contig can create significant ambiguities. This translates into either gaps in the sequence/physical map (DeSilva et al. 1999; Dunham et al. 1999) or the construction of physical maps in which BAC clones have been misassigned due to the presence of highly similar paralogous blocks (Cao et al. 1999). Indeed, in the recently reported DNA sequence of human chromosome 22 (Dunham et al. 1999), many of the gaps and map inconsistencies are biased toward the pericentromeric region at sites of large inter- and intrachromosomal repeats. The importance of such repeat elements in mediating recurrent chromosomal structural rearrangements (Ji et al. 1999; Mazzarella and Schlessinger 1998) and their overall abundance (∼10% based on chromosome 22 data) necessitate the development of specialized mapping and sequencing strategies to provide a comprehensive view of human genome organization. To circumvent some of the problems of paralogy, we developed a sequence-based strategy that exploits the paralogous nature of these complex regions. Our strategy is outlined in Figure ​Figure1.1. A completely sequenced clone from the pericentromeric region of 2p11 was chosen as a reference seed sequence. FISH analysis and database sequence similarity searches were used to provide a preliminary overview of the duplicative nature of this sequence. Next, a series of STS primer pairs were developed from the reference sequence and were used to screen a monochromosomal hybrid DNA panel by PCR. The STS product from each hybrid was directly sequenced, effectively generating a catalog of paralogous sequence variants that could be used to distinguish each chromosomal copy. The paralogous sequence variant is analogous to the single nucleotide polymorphism with the exception that sequence variation accumulates after a duplication event as opposed to descent from a common founding allele. Finally, the same STSs were used as probes to screen a human genomic BAC library. The BACs that positively hybridized were PCR amplified with the STS primer pairs and the products directly sequenced. Comparisons between the BAC sequence variants and monochromosomal paralogous sequence variants allowed us to unambiguously assign highly paralogous BAC clones to different chromosomal bins. This transchromosomal approach not only allows the rapid identification and characterization of other pericentromeric DNA but also provides insight into the unique structure and biology of these complex regions of the genome. Figure 1 Flowchart of pericentromeric characterization strategy.