Refinement of a chimpanzee pericentric inversion breakpoint to a segmental duplication cluster

Abstract

Background: Pericentric inversions are the most common euchromatic chromosomal differences among humans and the great apes. The human and chimpanzee karyotype differs by nine such events, in addition to several constitutive heterochromatic increases and one chromosomal fusion event. Reproductive isolation and subsequent speciation are thought to be the potential result of pericentric inversions, as reproductive boundaries form as a result of hybrid sterility.

Results: Here we employed a comparative fluorescence in situ hybridization approach, using probes selected from a combination of physical mapping, genomic sequence, and segmental duplication analyses to narrow the breakpoint interval of a pericentric inversion in chimpanzee involving the orthologous human 15q11-q13 region. We have refined the inversion breakpoint of this chimpanzee-specific rearrangement to a 600 kilobase (kb) interval of the human genome consisting of entirely duplicated material. Detailed analysis of the underlying sequence indicated that this region comprises multiple segmental duplications, including a previously characterized duplication of the alpha7 neuronal nicotinic acetylcholine receptor subunit gene (CHRNA7) in 15q13.3 and several Golgin-linked-to-PML, or LCR15, duplications.

Conclusions: We conclude that, on the basis of experimental data excluding the CHRNA7 duplicon as the site of inversion, and sequence analysis of regional duplications, the most likely rearrangement site is within a GLP/LCR15 duplicon. This study further exemplifies the genomic plasticity due to the presence of segmental duplications and highlights their importance for a complete understanding of genome evolution.

Figure 1

Map of Human 15q11-q13. Probes used to localize the chimpanzee (PTR) pericentric inversion are shown color-coded as to whether they hybridized to PTR XVp (yellow) or PTR XVq (blue). The breakpoint spanning clones are indicated in green. The three major PWS/AS common deletion breakpoints are indicated in red as BP1, BP2 and BP3. The previously characterized inv dup(15) breakpoint is indicated in orange as ID1. The duplicate CHRNA7 sequence is designated CHRNA7'. Accession numbers for all clones are indicated, all clones were obtained from the RPCI-11 BAC library. The map is not drawn to scale.

Figure 2

Pericentric inversion of HSA 15q11-q13 in chimpanzee two-color FISH. Probe RP11-88O16 (red), located in distal human 15q13.3, and probe pDJ-778A2 (green), which hybridizes to the HERC2-related PWS/AS breakpoint clusters in 15q11.2 and 15q13.1 clearly demonstrate the inversion of HSA 15q proximal material in chimpanzee (b). Humans (a) share the ancestral state with gorilla (c) and orangutan (d). The DAPI image for each chromosome is shown above the hybridization image and centromeres are indicated by the arrows.

Figure 3

FISH analysis of breakpoint-spanning clones. FISH hybridization results for probes spanning the PTR inversion breakpoint interval on human chromosome 15 (HSA) and chimpanzee chromosome XV (PTR) are shown. The DAPI image for each chromosome is shown above the hybridization image. PTR probes RP11-360J18 and RP11-736I24 flank the breakpoint interval on the PTR XVp and XVq side, respectively, and map uniquely. All clones tested that map between RP11-360J18 and RP11-736I24 demonstrated dual FISH signals on PTR XVp and XVq. The clones are ordered from left to right according to their starting position within the November 2002 human genome assembly (see Figure 4).

Figure 4

Segmental duplications in the inversion breakpoint interval. The human genomic sequence encompassing the most proximal PTR XVp probe (RP11-360J18) and most distal PTR XVq probe (RP11-736I24) was extracted from the genome assembly (November 2002) and sequence-similarity searches performed against the entire human genome (see Materials and methods). The output was displayed using the program Parasight (Jeff Bailey, unpublished work), which shows pairwise alignments as colored horizontal boxes below the heavy black line, which represents the 1 Mb interval. The color-coding of each horizontal box is a reflection of the sequence similarity of the alignment: 100-99% is indicated in red, 99-97% in orange, 97-95% in yellow, 95-93% in green, 93-91% in blue and 91-89% in purple. *Additional putative duplicons; these may, however, be artifacts of assembly and they require further verification.

Figure 5

Southern analysis of the CHRNA7 duplication. Probe CHRNA7-PstI was used to demonstrate a 104 bp RFLP distinguishing the intact (548 bp band) and duplicated (444 bp band) CHRNA7 loci in the human genome (lane 1). A single band - corresponding to the intact CHRNA7 locus - is seen in PTR, PPA and GGO (lanes 2 and 3, 4 and 5, 6 and 7, respectively), indicating that the duplication of CHRNA7 was a recent human-specific event and that therefore the gene is single copy in the PTR genome.