A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination

Abstract

Insights into the origins of structural variation and the mutational mechanisms underlying genomic disorders would be greatly improved by a genomewide map of hotspots of nonallelic homologous recombination (NAHR). Moreover, our understanding of sequence variation within the duplicated sequences that are substrates for NAHR lags far behind that of sequence variation within the single-copy portion of the genome. Perhaps the best-characterized NAHR hotspot lies within the 24-kb-long Charcot-Marie-Tooth disease type 1A (CMT1A)-repeats (REPs) that sponsor deletions and duplications that cause peripheral neuropathies. We investigated structural and sequence diversity within the CMT1A-REPs, both within and between species. We discovered a high frequency of retroelement insertions, accelerated sequence evolution after duplication, extensive paralogous gene conversion, and a greater than twofold enrichment of SNPs in humans relative to the genome average. We identified an allelic recombination hotspot underlying the known NAHR hotspot, which suggests that the two processes are intimately related. Finally, we used our data to develop a novel method for inferring the location of an NAHR hotspot from sequence variation within segmental duplications and applied it to identify a putative NAHR hotspot within the LCR22 repeats that sponsor velocardiofacial syndrome deletions. We propose that a large-scale project to map sequence variation within segmental duplications would reveal a wealth of novel chromosomal-rearrangement hotspots.

Figure  1.

Structural variation within CMT1A-REPs. Top panel shows the Southern probe binding site (horizontal shaded rectangle) and the positions of EcoRI sites in the CMT1A-REPs (black vertical bars). Middle panel shows the different Southern banding patterns identified in humans and chimpanzees, with each distinct pattern given a one-letter code. Bottom panel shows the sites of insertion events identified in the sequences described in the text. These insertion events are color coded, to identify which banding pattern they correspond with. A variable EcoRI restriction site is shown as a green bar, color coded to reflect the associated banding pattern. The absence of this restriction site, in combination with the presence of the partial (2,123-bp) L1PA2 insertion, accounts for the larger distal REP fragment apparent in chimpanzee Southern banding patterns F, G, and H, as compared to humans.

Figure  2.

Global Southern analysis of CMT1A-REPs. The figure displays a representative subset of the Southern banding patterns identified among humans in this study. Samples indicated in North America are from African Americans. Banding patterns that differ from the canonical two-band patterns seen in most humans are indicated with a black arrow.

Figure  3.

Phylogenies of CMT1A-REPs. Upper panel displays a rooted neighbor-joining phylogeny of CMT1A-REPs in hominoid species, labeled with bootstrap percentages. Proximal sequences are shown in red, and distal sequences in blue. Lower panel displays a SplitsTree of CMT1A-REPs in hominoid species, constructed using uncorrected P distances. Proximal sequences are shown in red, and distal sequences in blue.

Figure  4.

Definition and distribution of SNPs, PSVs, and MSVs. Upper panel shows examples of SNPs, PSVs, and MSVs within a portion of an alignment of three distal (gray) and three proximal (black) CMT1A-REP sequences. Lower panels display the positions of these three classes of variants within proximal and distal CMT1A-REPs in 20 human chromosomes.

Figure  5.

SplitsTree of all proximal (red) and distal (blue) human CMT1A-REPs

Figure  6.

Identifying NAHR hotspots from patterns of sequence variation. Upper panel shows how the concerted index varies along the length of a 24-kb alignment of human and chimpanzee CMT1A-REPs. Lower panel shows how an alternative sliding window statistic, the hotspot index, varies along an alignment of human proximal and distal CMT1A-REPs. Solid and dashed horizontal lines indicate the 95th and 99th percentiles, respectively, of the hotspot index, as determined by 10,000 random permutations of the positions of SNPs, PSVs, and MSVs.

Figure  7.

The hotspot index with potential CpG sites removed. This plot shows how the hotspot index varies along an alignment of proximal and distal CMT1A-REPs with MSVs at CpG sites removed from the data set (as described in the “Results” section). The solid and dashed horizontal lines indicate the 95th and 99th percentiles, respectively, of the hotspot index, as determined by 10,000 random permutations of the positions of SNPs, PSVs, and MSVs.

Figure  8.

Putative NAHR hotspots in LCR22-2 and LCR22-4. This figure shows how the hotspot index varies along an ∼180-kb alignment of LCR22-2 and LCR22-4. Solid and dashed horizontal lines indicate the 95th and 99th percentiles, respectively, of the hotspot index, as determined by 10,000 random permutations of the positions of SNPs, PSVs, and MSVs.

Figure  9.

Allelic recombination rates within CMT1A-REPs. Upper panel shows the estimated local recombination rates along proximal (gray) and distal (black) CMT1A-REPs with the use of all SNPs. Lower panel shows the estimated local recombination rates in proximal and distal CMT1A-REPs once SNPs in the known ∼700-bp hotspot (7,800–8,500 bp) have been removed. The location of the known NAHR hotspot is indicated.

Figure  10.

Hotspot index on thinned genotypic data. This plot shows how the hotspot index varies along an alignment of proximal and distal CMT1A-REPs with the data thinned (as described in the “Discussion” section) to include only those sites that could be known to be variant without resequencing. Solid and dashed horizontal lines indicate the 95th and 99th percentiles, respectively, of the hotspot index, as determined by 10,000 random permutations of the positions of SNPs, PSVs, and MSVs.