Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication

Abstract

Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology-or homeology-driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

Fig 1. Inversion discovery and frequency between A1a and A1b.

A) A depiction of the PLP1 genomic region on chromosome Xq22.2. PLP1 is proximal to the LCR structures and the black horizontal arrow indicates direction of transcription. There are three IRs present at chrX:103,171,387–103,359,682: the outer C and D repeats, middle A1a and A1b repeats, and inner A2 and A3 repeats [23,24]. The gene and the IR structures are separated by ~150 kb. B) A common inversion, discovered in the HGSV resource, is depicted between the A1a and A1b repeats. This inversion was present in at least 5/9 of individuals in the fosmid resource, and is possible in three additional people (S1 Fig). C) A Southern blotting scheme to distinguish between reference and inversion alleles is depicted, with A1a, A1b, A2 and A3 represented as above. Female genomes have two alleles, and phenotypically normal males have one allele (blot quantitated in S1 Table). Digestion with BssSI (depicted by black vertical lines) and detection with a probe proximal to LCR A1a (indicated by red star) should distinguish reference (25 kb, red) from inverted (29 kb, purple) alleles. Nine individuals from the HapMap population were studied for inversion via Southern blotting. Gender of the individual is indicated by circles (female) or squares (male) above the blot. DNA identifiers (NA numbers) are consistent with Coriell names (http://ccr.coriell.org/), and fosmid libraries (ABC library identifiers) are as in Kidd et al. [49]. The population of origin for each individual and the genotypes are indicated at the bottom of the blot. H = Han Chinese, J = Japanese, Y = Yoruban, and U = unknown. Genotyping for 8 additional individuals is depicted in S3 Fig.

Fig 2. Complex rearrangements involving PLP1 have clustered breakpoints in IRs.

A) 17 individuals are depicted with schematics of their array results. Duplications are indicated in red, triplications are indicated in blue, and the quadruplication is indicated in gold. Coordinates and LCR blocks are indicated in the cartoon above the array results (A2 and A3 are unlabeled). P1150 and P113 contain copy number neutral segments within the rearrangement, leading to a copy number of 1 indicated by white space in the array schematic (see also S5 and S6 Figs). B) FISH results for P642, depicting a large triplication of the PLP1 region, including the gene. All three copies of the four probes are on Xq22.2. Array data are at the top and qPCR data are below, with LCRs indicated in yellow and pink. Colors for copy number in array data are as in panel A. Probe locations for FISH denoted by vertical lines. C) FISH results for P1150, similar to those in B. The figure depicts duplication of the two proximal probes 1 and 2 and triplication of the distal 3 and 4 probes, as proposed by the array data above. D) FISH results for patient P113, depicting a quadruplication/duplication rearrangement of the region. High-resolution aCGH showed greater complexities, including a small triplication and a normal copy number segment within the duplication (See Figs. 5 and S5). In all FISH studies, nuclei are stained with DAPI, and a marker for the short arm of chromosome X, AL353698, is shown in green. Vertical arrows with numbers in the array data above indicate positions of FISH probes throughout the PLP1 region.

Fig 3. CGRs at the PLP1 locus use A1a and A1b repeats.

A) Southern scheme from Fig. 1 applied to a DUP-TRP/INV-DUP rearrangement results in either two copies of a 25 kb band and one of a 29 kb band if the rearrangement occurred on H1 (shown at the top), or the reciprocal copy dosage if the rearrangement occurred on H2 (bottom). Colors for LCRs are as in Fig. 1. B) Digested DNA from 2 control individuals (NA15510 and NA10851) and two PMD/SPG2 patients (BAB1290 and BAB1612/P374, respectively). Southern below depicts control individuals have expected, gender appropriate 2 and 1 copies, and affected males have three copies, with dosage of 2:1 H2:H1, indicating the rearrangement likely occurred on H2 (indicated within the black square for each patient- data for this blot and panels C and D are quantitated in S4 Table). C) BAB2389 and BAB3698 may also have rearrangements on the inverted allele (H2, indicated at the top of the image). BAB3698 is depicted with his carrier mother, sister (BAB3700 and BAB3699, indicated by a dot in a circle), and non-carrier grandmother (BAB4179). The grandfather was unaffected and unavailable for study. D) P250, P298, and P558 all likely contain rearrangements on H1 (~1:2 ratio of H2:H1) and P500, P518, and P642 contain rearrangements on H2. All rearrangement progenitor haplotypes are indicated for the patients above the Southern blot. E) The reference genomic structure of H1 is shown (inner A2 and A3 repeats are unlabeled). The qPCR primer pairs amplify a unique region outside of the A1a LCR (in red), inside of both A1a and A1b LCRs (in black) or from the A1a LCR to a unique region outside (red/black pair below). These will give rise to one copy (red pair and red/black pair) or two copies (black pair) in a non-rearranged X chromosome in a male individual. DUP-TRP/INV-DUP (on right) in an H1 haplotype will give rise to four copies amplified by the black pair (2x normal control) and three copies by the red pair and red/black pair (3X normal control) (S7 Fig). F) Analysis of Jct1 has successfully cloned one breakpoint in BAB1612/P374. The structure of the LCR-mediated rearrangement on H2 is depicted at the top (A1a and A1b are simplified to “A” and “B” and inner A2 and A3 repeats are unlabeled). Overlapping clones for each region of the two LCRs were generated (numbered 1–4, S8 Fig), and results for BAB1612/P374 were obtained for section 1 clones that both contain and lack the breakpoint. Multiple clones from this region are depicted along with the reference sequences for LCRs A1a and A1b below. The breakpoint from individual BAB1612/P374 occurred in stretch of 24bp of microhomology (bracket-denoted region).

Fig 4. DUP-TRP/INV-DUP structures at the PLP1 locus.

A) The aCGH results from patient P1407 showing a duplication of PLP1 and a distal triplication-duplication structure are shown at the top, with duplications in red and triplication in blue. The relative genomic regions are labeled with letters to distinguish their relative positions within the CGR. The IRs flanking segment e (A1a and A1b) are denoted by inverted blue and purple triangles, respectively. B) Two potential structures of the generalized DUP-TRP/INV-DUP rearrangement that are consistent with Jct2 sequencing are shown in the lower panel of the figure in relation to the canonical genomic structure at the top. Here, the unique proximal breakpoint junction location that differs between patients, the LCR-mediated distal inversion breakpoint junction, and the inverted triplication region are seen. Letters followed by a prime symbol indicate duplicated segments. Two prime symbols indicate the triplicated segment.

Fig 5. A quadruplication and potential rolling-circle mechanism of rearrangement.

A) P113 aCGH result is shown. At top, colored arrows represent segments of triplicated (blue), quadruplicated (gold), and duplicated (red) sequence in this individual. A1a and A1b are represented by inverted blue and purple triangles, respectively. Copy number (C#) of the various segments is enumerated below the aCGH result, as is the rearrangement structure on the H2 haplotype (see S9 Fig). Breakpoints are depicted by blue dashed arrows in the schematic of the rearrangement and are labeled with the template switching events associated with the junction. Breakpoint sequences are depicted in S6 Fig, and dPCR data indicating duplication of FoSTeS events 2 & 3 is in S5 Table. B) The proposed rolling circle mechanism leading to the triplication and quadruplication is depicted, with the FoSTeS events 2 & 3 leading to invasion of the fork into the already replicated strand. This establishes the rolling circle, depicted on the right. The event ends with a double strand break or a fork disassociation event, and the breakpoint involving FoSTeS event 1 (S6 Fig). The structure of the overall rearrangement is depicted at the bottom, with colors relating to panel A for orientation.