Uncommon deletions of the Smith-Magenis syndrome region can be recurrent when alternate low-copy repeats act as homologous recombination substrates

Abstract

Several homologous recombination "hotspots," or sites of positional preference for strand exchanges, associated with recurrent deletions and duplications have been reported within large low-copy repeats (LCRs). Recently, such a hotspot was identified in patients with the Smith-Magenis syndrome (SMS) common deletion of approximately 4 Mb or a reciprocal duplication within the KER gene cluster of the SMS-REP LCRs, in which 50% of analyzed strand exchanges resulting in deletion and 23% of those resulting in duplication occurred. Here, we report an additional recombination hotspot within LCR17pA and LCR17pD, which serve as alternative substrates for nonallelic homologous recombination that results in large (approximately 5 Mb) deletions of 17p11.2, which include the SMS region. Using polymerase-chain-reaction mapping of somatic cell hybrid lines, we refined the breakpoints of six deletions within these LCRs. Sequence analysis of the recombinant junctions revealed that all six strand exchanges occurred within a 524-bp interval, and four of them occurred within an AluSq/x element. This interval represents only 0.5% of the 124-kb stretch of 98.6% sequence identity between LCR17pA and LCR17pD. A search for potentially stimulating sequence motifs revealed short AT-rich segments flanking the recombination hotspot. Our findings indicate that alternative LCRs can mediate rearrangements, resulting in haploinsufficiency of the SMS critical region, and reimplicate homologous recombination as a major mechanism for genomic disorders.

Figure 1

Comparison of common and uncommon deletions of SMS. Proximal chromosome 17p is depicted at the bottom, with the position of LCRs shown. The common deletion is shown above, and the large deletions of SMS (discussed in the main text) are shown below. The solid horizontal lines represent the undeleted chromosome segments, and the dotted lines represent the deleted chromosome segments. The numbers identifying analyzed patients with large deletions are listed (right). The arrows indicate the distal and proximal breakpoints of the large deletions within LCR17pA and LCR17pD, respectively.

Figure 2

Detection of a patient-specific junction fragment. A, Schematic representation of the breakpoint regions in LCR17pA (white rectangle) and LCR17pD (gray rectangle). The dotted line represents the proposed crossover event between the LCRs; the 2.0-kb junction fragment results from this crossover. Small unblackened arrows denote the primers used for amplification of the 5.2-kb product containing the breakpoint region. AclI restriction sites and expected fragment sizes upon digestion of the PCR product are shown (not drawn to scale). B, Results of assay. The sizes of the bands are shown to the right; the patients and their parents are represented by the pedigrees at the top. All six patients, but none of the parents, have the expected 2.0-kb junction fragment, indicating that the six strand exchanges occurred within this interval. The 5.2-kb undigested fragment is visible and presumably a result of incomplete digestion. The 0.5-kb fragment from LCR17pA is not shown.

Figure 3

Fine mapping of the deletion breakpoints within the 2.0-kb AclI junction fragment. At the top is a diagram of the position of the hotspot (dotted box) within the LCR sequence, and a finer-scale view of the hotspot itself is shown with the telomere (tel) on the left and the centromere (cen) on the right. Paralogous sequence variants, or nucleotide cis-morphisms, between LCR17pA (above the line) and LCR17pD (below the line) are represented by uppercase letters. Polymorphic nucleotides are represented by lowercase letters. Restriction enzyme consensus sequence cis-morphisms (including the AclI sites used to isolate a patient-specific junction fragment) are also shown. Alu elements are depicted by the diagonally striped rectangles, and AT-rich segments are represented by the blackened boxes labeled A, B, or C (C represents the AT-rich segment distal to the hotspot, found only in LCR17pD). In the lower portion of the figure, patient numbers are given (right). Each circle represents a cis-morphic nucleotide within the patient’s recombinant LCR17pA/D. Unblackened circles denote nucleotides matching LCR17pA, and blackened circles denote nucleotides matching LCR17pD. Bold lines are drawn between the two cis-morphisms, in which the transition from distal-like to proximal-like sequence occurs for each patient. The sizes of these intervals are given at the bottom (not drawn to scale).