Smsreps Lcr Located At The Breakpoints Of The Common Deletion

Physical mapping studies have demonstrated that the SMS common deletion interval is flanked by large (approx 200 kb), highly identical (>98%), LCR gene clusters termed proximal and distal SMS-REPs (1,3,7) that during either maternal or paternal gametogenesis act as substrates for NAHR (8,25). To delineate the genomic structure (size, orientation, sequence identity, gene content) and evolution of the SMS-REPs, we constructed and sequenced a complete approx 5-Mb bacterial and P1-derived artificial chromosome (BAC/PAC) contig in 17p11.2-p12. Our analysis revealed that both the proximal SMS-REP (approx 256 kb) and the distal copy (approx 176 kb) are located in the same orientation and derived from a progenitor copy, whereas the middle SMS-REP (approx 241 kb) is inverted and appears to have been derived from the proximal copy. This architecture likely explains why the common SMS deletions occur between proximal and distal SMS-REPs.

There are four regions of significant stretches of identity between the proximal and the distal SMS-REPs (A, B, C, and D regions in Fig. 1A). The sum of these high sequence similarity regions is approx 170 kb (169,905 bp), and the identity is greater than 98% with the exception of the D region (>95%) (Table 1). The largest conserved segment (region A in Fig. 1A) is 126 kb in size. Two large sequence blocks (between A and B, and between C and D) in the proximal SMS-REP are absent in the distal SMS-REP. Two smaller blocks, flanking areas of the B region in the distal SMS-REP are absent in the proximal SMS-REP (Fig. 1A).

FISH analysis using SMS-REP-specific BAC clones as probes revealed strong hybridization signals on metaphase chromosomes 17p11.2 and three strong signals on the interphase chromosomes. However, SMS-REP-specific BACs also showed weaker hybridization signals in interphase analysis and metaphase spreads; these map to chromosome 17p13.1, 17p12,

Fig. 1. Sequence-based genomic structure of the Smith-Magenis syndrome (SMS)-REPs. (A) There are four regions of sequence identity more than 95% between the proximal and the distal SMS-REPs (A, B, C, and D). The A, B, and C sequence blocks have more than 98% identity between distal and proximal REPs, whereas the D regions (green) show more than 95% identity. The thick blue lines represent the

Fig. 1. Sequence-based genomic structure of the Smith-Magenis syndrome (SMS)-REPs. (A) There are four regions of sequence identity more than 95% between the proximal and the distal SMS-REPs (A, B, C, and D). The A, B, and C sequence blocks have more than 98% identity between distal and proximal REPs, whereas the D regions (green) show more than 95% identity. The thick blue lines represent the

17q11.2, 17q12, 17q21.2, and 17q23.2. In concordance with FISH results, BLAST analysis revealed that other approx 11-30 kb "SMS-REP-like" paralogous sequences, fragments of SMS-REPs, are localized on 17p13.1 (approx 28 kb), 17p12 (approx 11 kb), 17q11.2 (approx 30 kb), 17q12 (approx 11 kb), 17q21.2 (approx 25 kb), and 17q23.2 (approx 28 kb) (7).

Sequence analyses show that all three SMS-REPs within the SMS common deletion are not present in the mouse syntenic region (16). Apparently, except for a chromosome inversion of the region between the middle and proximal SMS-REP syntenic region in mouse, the gene order between SMS-REPs is conserved (16,26). Interestingly, transposition occurred for the TACI and KCNJ12 genes adjacent to the SMS-REPs. This rearrangement of gene order might have occurred during the evolution of the SMS-REPs, indicating that segmental duplications might transpose surrounding genes (16).

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