Inversion Polymorphism Discovery, Frequency and Recurrence

The 186 kb genomic interval (ChrX: 103,172,000–103,358,000 in hg19) located ~150 kb distal to PLP1 contains a complex genomic architecture in the haploid reference genome. This region consists of an array of IRs, with the ~40 kb outer C and D repeats having ~93% identity, the middle A1a and A1b repeats ~20 kb in size and ~99% identical, and the innermost ~10 kb A2 and A3 repeats showing ~87% identity both with each other and with A1a and A1b (Fig. 1A) [16,23,24]. The IR architecture predicts the potential for inversion mediated by non-allelic homologous recombination (NAHR), resulting in at least two structural haplotypes, analogous to the H1 and H2 structural variant (SV) alleles at the MECP2 locus [1]. Indeed, in silico analysis of the human genome SV track from the UCSC Genome Browser (www.genome.ucsc.edu) suggests the existence of such an SV allele [25]. The browser track indicates fosmids consistent with inversions spanning both of the A1a and A1b LCRs in 5 of 9 individuals (S1 Fig) [8,26]. These data indicate that there was an inversion between A1a and A1b LCRs and that the inversion haplotype exists at a relatively high allele frequency as a non-pathogenic rearrangement in HapMap individuals (Fig. 1B). Further investigation mapped the apparent ectopic crossover for the NAHR-mediated inversion in a fosmid from the G248 library to nucleotide-level resolution (S2 Fig).

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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.

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To directly examine the inversion SV polymorphism between A1a and A1b, we designed a Southern blotting assay and genotyped multiple individuals from different populations of origin for reference (arbitrarily designated H1) or inversion (H2) structural haplotypes. The scheme of the assay is depicted in Fig. 1C, wherein Southern analysis leads to predicted visible fragments of 25 kb for H1 and/or 29 kb for H2. As the rearrangement is on the X chromosome, males should have only one allele, and females, two. Genotyping 17 individuals (including 3 males) with this assay discerned 31 haplotypes of the X chromosome (Figs. 1C, S3, and S1 Table). The frequencies of structural haplotypes were 13/31 H2 (~42%) and 18/31 H1 (~58%), with 4 individuals hemizygous or homozygous for H2 and 7 for H1. The remaining 6 females were heterozygous for both H1 and H2. The 17 individuals were of Japanese, CEPH Northern European, Han Chinese, Yoruban and unknown populations of origin, and all populations contained both H1 and H2 structural haplotypes (S2 Table).

We hypothesized that the similarity between LCRs A1a and A1b and their relatively large length and proximity (~20 kb repeats of ~99% identity separated by ~50 kb) could predispose to recurrent events [27,28]. We analyzed the genomic region encompassing the two LCRs and identified multiple adjacent single nucleotide polymorphisms (SNPs) spanning the region in linkage disequilibrium and delineating a haplotype block extending for ~0.5 Mb with a recombination rate of 0.3 centimorgans per Mb. The two SNP haplotype blocks were evenly distributed between the 14 different populations from the 1,000 genomes project [29]. Superimposing Southern blotting results for individuals homozygous or hemizygous for SV haplotypes on top of the SNP haplotypes enabled phasing; 6/7 inversion (H2) alleles were on one SNP haplotype and 1/7 was on the other (belonging to individual NA18947, S4 Fig), whereas homozygous H1 alleles occurred on either SNP haplotype. Heterozygous calls are uninformative, as the structural haplotype information cannot be phased to the SNP data. These data suggest that the inversion is likely recurrent in the population and makes population estimation of the structural variant using SNP genotyping unlikely to reflect the true population frequency.

Breakpoint Mapping of PLP1 Rearrangements

Sixteen patients with PMD and one diagnosed with spastic paraplegia type two (SPG2; MIM#312920) were examined by aCGH for copy number variation (CNV) in PLP1 and the surrounding genomic region. A schematic of CNV observed in the personal genomes from 17 patients is depicted in Fig. 2A. PLP1 duplications were detected in 10 patients (BAB1290, BAB2389, P250, P298, P500, P558, P842, P1389, P1407 and P113), whereas triplications were detected in 6 patients (BAB3698, P518, P642, P674, P820, and P1150). The one SPG2 patient, BAB1612/P374 has been described previously, and the phenotype of this individual is ascribed to a potential position effect [30]. The distal breakpoints in all subjects appear to cluster in approximately the same genomic location; however, there are few probes on the arrays that can specify unique loci within the C/D, A1a/A1b, and A2/A3 LCRs due to the repeat nature of the region. Thus, determining the precise LCRs involved in the breakpoints required alternate mapping approaches.

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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.

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Array and semi-quantitative PCR data, summarized in Fig. 2A (see also S5 Fig and Table 1), indicate that the region of rearrangement spans from 145 kb (BAB1612/P374) to ~4,000 kb (BAB2389). Triplicated genomic segments range in size from 254 bp (P298/P255) to 575 kb (P642). Proximal triplication and duplication copy number transitions differed in each individual and were not located within LCRs. The distal copy number transitions group within a 100 kb region of uncertainty as described above. The triplication present in P298/P255 was too small to be detected by aCGH; however, a 254 bp triplicated genomic segment was detected both by amplification and sequence analysis with unique flanking primers and by quantitative PCR (S6 Fig).

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Table 1. Unique Junctions (Jct2) in DUP-TRP/INV-DUP rearrangements.

https://doi.org/10.1371/journal.pgen.1005050.t001

FISH was performed on nuclei prepared from peripheral blood lymphocytes from P642, P1150, and P113. This independently corroborated interpretation of array and semi-quantitative PCR data using an orthogonal experimental approach. Moreover, FISH determined whether extra copies of the genomic segments were located in or near the PLP1 locus as opposed to elsewhere in the genome; arrays determine neither the orientation nor the position of a copy number segment, but only specify the genomic segment that underwent a gain in copy number (Fig. 2B-D). Interphase nuclei of patients P642 and P1150 showed, as expected, one green control probe signal and revealed three closely-spaced red PLP1 probe signals indicating triplication at the PLP1 locus; P113 had two red PLP1 signals indicating duplication at that locus, but four proximal probe signals confirmed the additional quadruplication (Fig. 2D). Metaphase spreads of all 3 patients gave one green control probe signal, one red presumably merged PLP1 probe signal on the X chromosome, and no signals on other chromosomes, also indicating that the triplications and duplications were at the PLP1 locus rather than being located elsewhere in the genome and that the triplications were too small to resolve on metaphase chromosomes.

Marker Genotypes Suggest Intra-Chromosomal Events

We investigated haplotypes using genetic markers, 2 short tandem repeats (STRs) and 9 SNPs, mapping over a 258 kb region of the duplications with 4 markers mapping within PLP1 and the remainder distal to it. We observed that 12 of 13 patients tested (P250, P255/298, BAB1612/P374, P500, P518, P558, P642, P674, P820, P842, P1150, P1389, and P1407), were monomorphic displaying only one form for each marker genotype (S3 Table). The DNA from BAB1612/P374 was only interrogated at the 7 sites distal to PLP1, since this is where his triplicated/duplicated region lies. In this subject, only one form was detected for all markers except the STR furthest distal to PLP1 where two were detected. The finding of an absence of bi-allelic loci in these multi-copy regions of X is most parsimoniously explained by the occurrence of intra-chromosomal rearrangement events, as has also been observed for DUP-TRP/INV-DUP rearrangements at the MECP2 locus [20].

Junction Analyses of Proximal Breakpoints

In P1150, we obtained a breakpoint junction between the proximal (centromeric) endpoint of the rearrangement and the proximal end of the triplicated region via inverse PCR. We then hypothesized that our other patients with duplication-triplication-duplication copy number changes could potentially have the same CGR product structure and explored this hypothesis by long-range PCR on each individual personal genome. We were able to amplify and sequence across the proximal breakpoint junction in all 16 patients (Table 1). The 16 junctions each indicate that the triplicated region is inverted with respect to the proximal duplication region (S6 Fig); in 6 cases the triplication encompasses PLP1. This is a potentially analogous rearrangement structure to that previously described for MECP2 CGRs; therefore, we denote the non-recurrent junctions in Table 1 as Jct2 [1].

We had previously mapped and sequenced across the duplication breakpoint junction of patient P255, who had a 254 bp inverted duplication [24]. We no longer had DNA from P255 to interrogate the copy number of the region; therefore, we tested an affected family member, P298, by qPCR and found, as anticipated, that the region is triplicated.

The Jct2 sequences in the 16 patients are shown in Table 1. Fourteen patients contain one or more breakpoint junctions displaying microhomology. Patients P558 and P842 have blunt junctions. In 13 of the patients, endpoints at Jct2 are in repetitive element sequences, and in P1389, one end was in an LCR (Table 1). Patient BAB1612/P374 contained a LINE2-mediated event (L2/L2, both within the same LCR) that did not result in a chimeric element. Patients P518 and BAB3698 contain chimeric AluS elements formed in the generation of this junction. In BAB3698, there are 47 bp of identity between the two AluSx elements at the transition from triplication to duplication. In P518, the rearrangement occurs through the formation of two AluS chimeric junctions, the first (from proximal to middle segment) in the same orientation in 14 bp of identity, and the second (from middle segment to triplication) in 34 bp of identity (see S6 Fig). This complex breakpoint junction contains a segment of 488 bp that consists of an AluSx3, an L1 sequence (L1ME), and an AluSq2. Interestingly, the distal (triplication) to middle junction occurs between these two Alu elements that are only separated by 310 bp, suggesting a potential U-turn caused by inverted Alus within close proximity [31,32], similar to the situation in Jct1 but mediated by short Alu sequences instead of LCRs (S6 Fig). The breakpoint junction mutational signatures are consistent with replicative mechanisms such as MMBIR or a homeologous (near homologous) recombination event between similar Alu elements at each instance of Jct2 [22,33,34].

Complexities that included an additional template switch were observed in Jct2 from individuals P500, P518, BAB1290 and BAB2389 (Table 1 and S6 Fig). Such events have been postulated to reflect reduced processivity of the replisome mediating MMBIR during initial template switching [22]. We also amplified across a breakpoint junction present in P1150, indicating a 27 kb deletion on one of the duplicated copies (Figs. 2 and S5). At that junction, there is a bp of microhomology (S6 Fig). The overall findings for Jct2 are consistent with both long distance template switching and a microhomology-mediated mechanistic process such as FoSTeS/MMBIR [21,22].

DUP-TRP/INV-DUP Distal Breakpoints

After Jct2 was determined for the 16 patients, we hypothesized that a likely genomic arrangement consistent with this junction was one in which one copy of the triplicated region was situated in an inverse orientation between the two copies of the duplicated region and that the other two copies of the triplication were embedded within the duplicated regions, i.e. a DUP-TRP/INV-DUP structure [1].

Patients with presumed DUP-TRP/INV-DUP rearrangements with sufficient DNA available were subjected to Southern blotting (10/16 total) to examine whether the same repeats involved in the common inversion polymorphism are also involved in the CGR and to investigate on which structural haplotype the rearrangement occurred. The Southern scheme in Fig. 1C was used to analyze patient DNAs; however, in a male with PMD caused by DUP-TRP/INV-DUP involving the A1a and A1b repeats, the Southern blot does not reflect the normal copy number of one allele of the X chromosome (either H1 or H2) (Fig. 3A, S4 Table). Instead, the rearrangement gives rise to two copies of the original haplotype plus an additional “flipped” haplotype in an affected individual with DUP-TRP/INV-DUP leading to PMD, similar to the observation described for the MECP2 locus [1]. This assay can presumably distinguish the SV haplotype on which the genomic rearrangement occurred. A representative gel and labeled blot are shown in Fig. 3B, with the dosage of the bands indicating that subjects BAB1290 and BAB1612/P374 both carried the inversion H2 structural haplotype prior to the rearrangement.

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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).

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Interestingly, the 10 individuals examined by this assay appeared to use the A1a and A1b LCRs as the substrates for their rearrangements, in spite of two other IRs being located in close proximity (BIR between C/D would lead to duplication of H1 or H2 and A2/A3 would lead to triplication) (Figs. 1A, 3C and D). Individuals BAB1290, BAB1612/P374, BAB2389, BAB3698, P500, P518, and P642 all contained a rearrangement that had occurred on the inverted H2 allele, while P250, P298, and P558 had a Southern blot result indicating the rearrangement occurred on an H1 haplotype (S4 Table).

A three-generation family was studied in which the two maternal grandparents were unaffected, and subsequent Southern blotting and aCGH data indicated that the grandmother (BAB4179) was not a carrier and that she had two copies of the inverted H2 locus (Figs. 3C and S5). The grandfather was unavailable, but did not have PMD; therefore, the de novo rearrangement can be inferred to have occurred in between the grandparent and the maternal generation. The mother (BAB3700) was a carrier of the rearrangement and had equal dosage of H1 and H2 on a Southern Blot. The affected son (BAB3698) had Southern results consistent with rearrangement on H2, and his carrier sister (BAB3699) had similar results to the mother. These findings are consistent with the de novo DUP-TRP/INV-DUP occurring in association with “flipping” the H2 haplotype to an H1 haplotype, a mechanism similar to that observed for CGRs at the MECP2 locus (Fig. 3A) [1]. The assay results in this family are most parsimonious with the rearrangement occurring on one of the grandmother’s inversion-containing alleles (H2), and having balanced copy number in BAB3700 and BAB3699 due to the additional allele being a reference (H1) 25 kb band. This would result in a 2:2 dosage of 29 kb:25 kb bands on the Southern blot, which we observe in both BAB3700 and 3699 (Fig. 3C and S4 Table).

Breakpoint Junctions or Crossovers Within LCRs

As Jct1 occurs within the LCR region distal to PLP1, the junctional products are not readily amplified and sequenced by long PCR with primers anchored to unique flanking sequence. We adopted an alternative strategy to complement the Southern blotting assay above. Using a semi-quantitative PCR approach, we first confirmed that each of the patients has duplication of A1a and A1b LCRs (black primer pair in Fig. 3E) and triplication of a region proximal to A1a (red primer pair and black/red primer pair in Figs. 3E, S7). This PCR approach independently verified the Southern Blot results and suggested a crossover breakpoint within the A1a or A1a/A1b chimera present on H2 (Fig. 3E). We attempted to more narrowly define the crossover region in our patients by using sequence differences between the LCRs (paralogous sequence variants or PSVs), but patients appeared to lack apparent PSVs between A1a and A1b that were at the corresponding genomic locations in the hg19 reference sequence [35].

To determine sequences across Jct1, we designed a PCR-cloning assay that allowed us to amplify large (~12–16 kb), overlapping portions of both A1a and A1b LCRs that are implicated in the rearrangements [35] (Figs. 3F, S8). Three individuals were subjected to this analysis (BAB1612/P374, BAB2389, and BAB1290), however BAB2389 and BAB1290 appear to have Jct1 within a large region of identity (>8 kb) in the center of the LCR that lacks PSVs between cloned segments; therefore, further refinement of the breakpoint junction was intractable using this method. Additionally, in P255/298, a PCR approach using one primer at the proximal duplication junction and one within the LCR corroborated that the breakpoint indeed occurred within this >8kb stretch of identity.

In contrast to the three other individuals for whom we sought to find Jct1 at base pair resolution, in BAB1612/P374 we were able to detect an LCR-mediated breakpoint within 24 bp of microhomology flanked by A1a and A1b sequences (Fig. 3F). The point of crossover within this sequence was confirmed by direct PCR amplification and sequence analysis from genomic DNA followed by comparison to the PSVs present on cloned A1a and A1b sequences from the same individual; its identification elucidates Jct1 within an LCR, a heretofore un-investigated junction at the nucleotide level of resolution.

DUP-TRP/INV-DUP Rearrangement Structure

The DUP-TRP/INV-DUP structure hypothesized for these 16 individuals postulates that although there are 4 copy number transitions in these patients, there are only two breakpoint junctions (Fig. 4A). We have sequenced Jct2 in all 16 patients; Southern blotting and quantitative PCR were used to determine Jct1, and direct junction sequencing was successful for BAB1612/P374 (Figs. 3, 4 and S6). Additionally, due to the small size (~ 254 bp) of the triplication in P255/298, a PCR approach using one primer at the proximal duplication junction and one within the LCR validated the overall structure of this rearrangement as DUP-TRP/INV-DUP.

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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.

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Quadruplication by Rolling-Circle Amplification

We have discerned two junctions from patient P113 with proximal quadruplication and duplication of PLP1 using long-range PCR (Figs. 5A, S6). Junction 1 consists of one fork stalling and template switching (FoSTeS) event—FoSTeS 1 (Fig. 5A). The second junction, between the proximal end of the triplication and the distal end of the quadruplication, consists of FoSTeS events 2 and 3 (S6 Fig for sequences of all junctions). We determined that the rearrangement was on the inverted H2 allele using PCR genotyping of the haplotype present in P113 (S9 Fig). Additionally, digital PCR (dPCR) data indicate that the FoSTeS 1 occurs in one copy, and FoSTeS 2/3 occurs in 2 copies (S5 Table).

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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.

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This quadruplication rearrangement is also associated with a de novo point mutation (G insertion) ~50 bp away from the junction that appeared to occur concurrent with the rearrangement, as observed for other CGR mediated by a replicative process (S6 Fig) [20]. The mechanism by which copy number increased from 3 to 4 copies and generated the quadruplication is suggestive of a rolling circle amplification, wherein one breakpoint is repeated twice in the process of replicating ~280 kb (S5 Table) [22,36–38]. The FISH data for this individual shows the rearrangement to be contained on the X chromosome, and family data including the proband P113, his mother P154, his affected uncle P117, and grandmother P088 suggest that the structure is stable in 4 individuals from 3 generations, diminishing the likelihood of recombination-based amplification (Figs. 2D and S5). If the amplification were mediated by NAHR (see S10 Fig), this rearrangement would contain two templates for subsequent rounds of amplification. Therefore, the rearrangement in P113 should be twice as likely to undergo expansion as the proposed intermediate. Additionally, the presence of monomorphic SNPs throughout the region of copy number gains on SNP arrays indicates that the rearrangement was intra-chromosomal, as was found for the 13 patients with DUP-TRP/INV-DUP rearrangements (S11 Fig). The quadruplication-containing CGR was observed to transmit stably and co-segregate with disease through three generation (S5 and S11 Figs). A proposed mechanism for the rearrangement occurring in one complex quadruplication event is shown in Fig. 5 and consists of a rolling-circle amplification of the triplicated and quadruplicated segments.

Inversion Ascertainment

To determine whether there is a polymorphic inversion in the LCRs distal to PLP1, we examined the genomic information for 9 individuals contained in the human genome structural variation (HGSV) track of the UCSC Genome Browser [8,25,26]. The HGSV track (hg18) contains data on discordant fosmid end sequences from libraries of 9 individuals from diverse geographical regions. Discordant end sequence orientations of fosmids spanning LCRs A1a or A1b [23] and having both ends present in unique sequence (not LCRs) indicate potential inversions [26]. Individuals with at least one clone independently spanning each of the LCRs suggest that there is an inversion between the two repeats (S1 Fig).

Inversion Haplotypes and Analysis

Phased data from the 1000 genomes project [29] was used to create plots of two haplotypes in the region spanning from LCRs A1a to A1b (Hg19 coordinates, ChrX:103223669–103324337) [48]. One thousand genomes data was cross-correlated with homozygous genotypes determined from Southern Blots to elucidate phased haplotypes that contain inversion alleles. Results were plotted using custom (in-house) scripts implemented in the R programming language (S4 Fig).

Personal Genomes from Subjects and Patients Investigated

Families with PMD or rearrangements of Xq22.2 including PLP1 were obtained by physician referral or self-referral. Patients were enrolled through informed consent in research protocols approved by the Institutional Review Boards at Baylor College of Medicine (BCM) and the Nemours Alfred I. duPont Hospital for Children. The rearrangements present in patients BAB1290, BAB1612/P374, BAB2389, P250, P255, P500, P518, and P558 were published previously [1,24,30]. Two of the patients with PLP1 triplication (P518 and P674) were described as having more severe disease than patients with duplication [14]. Control DNAs from HapMap individuals [48] were obtained from the Coriell Institute for Medical Research cell repositories.

DNA Digestion and Fragment Separation for Southern Blotting Analysis

Approximately 10 μg of genomic DNA from each patient was digested using BssSI. The DNA was diluted to 60 μl and digested for 4 hours at 37°C with 16U, heat inactivated at 80°C for 20 minutes, and the digest was then repeated with 12U for 3 hours and subsequent heat inactivation (leading to a 10-fold overdigestion). The digested DNAs were then precipitated and concentrated using standard sodium acetate precipitation, and were reconstituted in 25 μl of water with gentle mixing overnight. Concentrations were determined using a NanoDrop spectrophotometer, and samples were then loaded along with an 8–48 kb ladder on a 0.6% Tris- Boric Acid-EDTA (TBE) gel and run in 1X TBE buffer for ~3 days at 50–60 volts. DNA restriction digestion products, i.e. bands on gels, were then visualized with ethidium bromide staining.

Probe Design for Southern Blot Analyses

Probe DNA was prepared using primers A1a proximal probe For- 5′-AATGCAGCTCAAAGGAAAGC-3′ and A1a proximal probe Rev- 5′-AGCCACTGACCAGTGATTTTC-3′ and amplifying a 514 bp fragment from BAC clone RP11–462K21 (https://bacpac.chori.org) DNA prepared using a QIAprep spin miniprep kit. The resultant PCR bands were resolved on 1% agarose and Tris-Acetate-EDTA gels and purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research). Probe DNA was validated by Sanger sequencing, using both forward and reverse primers and was frozen at-20°C in 90 ng aliquots.

Southern Blotting

Southern Blotting was carried out as previously described [23]. Briefly, DNAs were subjected to electrophoresis for sufficient duration to distinguish 25 and 29 kb fragment sizes and were then transferred to a Sure Blot positively charged nylon membrane by standard ‘sandwich’ methodology for 2–3 days. Approximately 80 ng of DNA was labeled with ³²P-dCTP by random priming for 2–4 hours at 37°C using the Random Primed DNA labeling kit (Roche). Membranes were pre-hybridized for 4 hours in 10% dextran sulfate/1M NaCl/1%SDS (hybridization solution) with 4mg of sheared salmon sperm DNA at 65°C. Probe was pre-associated in hybridization solution with ~1mg sheared placental DNA at 65°C for ~2 hours, then added to the pre-hybridized membrane. Hybridization was carried out at 65°C overnight (~18 hours). The following day, the blot was washed and analyzed using autoradiography for bands corresponding to PLP1 A1a structural haplotype information (~25 and 29 kb).

Array CGH Analyses

To determine the size, genomic content, and extent of PLP1 rearrangements, a high-density oligonucleotide array from Agilent was custom-designed to examine PMD patients. The 4 x 44 K microarray was designed using the Agilent eArray website (https://earray.chem.agilent.com/earray/) and was used to visualize the rearrangements of three patients in this study (BAB1290, BAB1612/P374, and BAB2389), the family containing individuals BAB3698, BAB3699, BAB3700, and BAB4179, and to complement existing array data for P500, P1407, and P113. The family of P113 was explored using Agilent arrays, including patients P113 and P117, as well as the mother of P113, P154, and grandmother, P088, who are both carriers. Probe labeling and hybridization were conducted as previously described, with NA15510 and NA10851 used as reference DNAs for female and male individuals, respectively (Accession GSE63594) [1]. Purified DNA samples from P113, P250, P500, P518, P558, P642, P674, P820, P842 and P1150 were submitted to NimbleGen for array service with normal male control NM002 as a reference. The NimbleGen X chromosome CGH fine-tiling array with oligonucleotide probes of 45 to 85 bases in length with median spacing of 106 bp throughout the whole X chromosome was used. Patient DNA samples P255/P298, BAB1612/P374, P1389 and P1407 were submitted to the Biomolecular Core Lab at duPont Hospital for Children for hybridization to Affymetrix Cytogenetics 2.7M Array. DNA sample P1407 was submitted to Coriell’s Genotyping and Microarray Center for hybridization on Affymetrix Genome Wide Human SNP Array 6.0. All data from Affymetrix arrays were analyzed with GeneChip Command Console Software AGCC. NimbleGen and Affymetrix Cytogenetics array data were aligned with qPCR data and plotted using the R programming language (Affymetrix and NimbleGen data are under Accession GSE64122) (Figs. 2B, C, D and S5).

Semi-Quantitative Multiplex PCR (qPCR) for Detection of Copy Number

Semi-quantitative multiplex PCR was performed using a QIAGEN Multiplex PCR kit according to the manufacturer’s protocol to analyze regions on the X chromosome in and surrounding PLP1 to determine copy number. Primer pairs were selected using the NCBI primer design tool (primers available upon request). In each experiment, five control DNAs were used, two known to have duplications in the region of interest without CGRs and three normal controls known to be single copy in the region of interest. A primer pair that amplifies a region of the human dystrophin (DMD) gene on the p-arm of the X chromosome was included in each multiplex reaction for amplification of a single-copy region. Products were separated by electrophoresis on a 4% NuSieve 3:1 agarose gel (Lonza, Walkersville MD) and stained with ethidium bromide. Net intensity of each band was determined using a Molecular Imaging system with Kodak Gel Logic Imaging software or AlphaImager HP. Copy number was determined by calculating the ratio of the net intensities of bands in the test region to dystrophin single-copy region for each DNA sample and then normalized by dividing by the average of the ratios of test region to dystrophin of the three normal controls. Theoretical ratios were: one, single-copy; two, duplication; three, triplication. Alternatively, quantitative PCR was performed as above except that one primer of each pair was labeled with 6-FAM and samples were submitted to the Biomolecular Core Lab at duPont Hospital for Children for capillary electrophoresis on an ABI PRISM 3130XL DNA Analyzer. Analysis of copy number was determined as above, by using area under the peak as determined by Peak Scanner software rather than net intensity. Triplicated, quadruplicated and duplicated regions were mapped to within several kb of their endpoints using these methods.

Fluorescent in situ Hybridization (FISH) to Confirm Copy Number

Interphase nuclei and metaphase chromosomes were prepared from 700 μl of whole blood stored in sodium heparin Vacutainer tubes as follows. Blood was placed in α-MEM, 20%FBS, 1% L-glutamine, 50 μg/ml gentamycin and treated with 150 μl Phytohemagluttenin (Invitrogen, Carlsbad CA). The cultures were incubated at 37°C for 72 hours in upright position after which they were treated with 100 μl colcemid by trituration followed by incubation at 37°C for 30 min. Cultures were then subjected to centrifugation at 350 x g for 6 minutes. The supernatant liquid was discarded and the pellet was suspended in 10 ml 75mM KCl pre-warmed to 37°C and incubated at 37°C for 15 minutes. Then 1 ml of fixative (3:1 mixture of methanol:acetic acid) was added slowly. The preparation was washed 3 times in 10 ml of fixative with pelleting by centrifugation at 350xg for 6 minutes after washes. The resulting pellet of interphase and metaphase chromosomes was then stored in fixative at -20°C. Chromosomes and nuclei were dropped onto pre-cleaned Fisherbrand slides in a CDS-5 Glovebox environmental Chamber, (Thermotron, Holland Michigan) set at 25°C and 50% humidity. Slides were stored at -20°C in a vacuum under dessication until use.

FISH was performed using cosmid clone U125A1 and BAC clone RP13–188A5 obtained from the BACPAC resource center. Cosmid and BAC DNAs were isolated using the QIAGEN Plasmid purification and QIAGEN Large-Construct kits, respectively. One μg of U125A1 DNA was labeled with Biotin-16-dUTP and 1μg RP13–188A5 DNA was labeled with digoxigenin using the DIG-Nick Translation Mix. Labeled probes were purified using Nuctrap probe purification columns according to the manufacturer’s protocol. After hybridization to chromosomes and nuclei according to standard protocol, biotinylated U125A1 was bound to Cy3-labeled streptavidin, further amplified with biotinylated antiavidin (Vector Laboratories, Burlingame CA) and detected with a second layer of Cy3-labeled streptavidin. Simultaneously, RP13–188A5 labeled with digoxigenin was coupled with mouse antidigoxigenin, detected with rabbit anti-mouse FITC (Jackson ImmunoResearch Laboratories) and further amplified with goat anti-rabbit FITC antibody. Nuclei and chromosome spreads were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and cover slips were mounted using Vectashield antifade solution. Images were captured using a Leica DM RXA2 fluorescence microscope and Openlab imaging software (Perkin Elmer, Waltham MA).

STR and SNP Analyses to Determine Origin of Extra Genomic Copies

To examine whether an intra- or inter-chromosomal origin occurred for the extra genomic segments in each patient’s genome, we analyzed 2 STRs and 9 single SNPs within the duplicated/triplicated region common to most patients. Sites were chosen based on marker genotypes displaying a high degree of heterozygosity in HapMap samples. S6 Table depicts the dbSNP identifiers, locations with respect to Chromosome X sequence NT_011651.17, and primers used to amplify the SNP or STR. Regions of interest were amplified from DNA with HotStar Taq DNA polymerase (Qiagen) for products <1kb or Expand High Fidelity PCR system (Roche) for products >1kb. Patients P250, P255/298, BAB1612/P374, P500, P518, P558, P642, P674, P820, P842, P1150, P1389, and P1407 were analyzed. Products containing the STR were amplified with one primer within the pair fluorescently-labeled so product sizes could be evaluated by capillary electrophoresis on ABI’s PRISM 3130 XL DNA Analyzer. Products containing the SNP were purified using QIAquick PCR or Gel purification kits, then sequenced with the Big Dye Terminator kit v. 3.1 (Life Technologies), according to the manufacturer’s instructions. Patients P113, P117 and carriers P154 and P088 were subjected to genotyping using the Illumina OmniExpress SNP array analyses at the human genome sequencing center of BCM. Data from the analyses was visualized by plotting the B allele frequencies versus the X chromosome coordinates encompassing the quadruplication genomic rearrangement, as well as the Log ratio of SNP intensity (S11 Fig).

Jct2 Analyses

Inverse PCR was used to obtain the first junction of patient P1150. Briefly, DNA was digested with NheI and ligated to form circles. PCR primers were designed to amplify in opposite directions around the circle by long-range PCR using the Expand High Fidelity PCR system (S6 Table). When the PCR products were analyzed on an agarose gel, a product was found that was unique to the patient. The product was subjected to DNA sequencing according to the manufacturer’s instructions, then purified with the Filtration Cartridge (Edge Biosystems, Inc., Gaithersburg MD) and separated using an ABI PRISM 3130 xl Genetic Analyzer. DNA sequence was analyzed using Vector NTI sequence analysis software.

Proximal junctions were obtained for the personal genomes from the remaining triplication patients by long-range PCR using appropriately positioned primers at the endpoints of copy number changes (Table 1 and S6 Table) in 25 μl reactions with 50–100 ng of patient DNA using TaKaRa LA Taq or using the Expand High Fidelity PCR dNTPack kit according to the manufacturers’ instructions. PCR products were prepared for sequencing by using the standard ExoSAP-IT protocol (Affymetrix, Santa Clara CA) or by using the Qiagen PCR purification kit and DNA sequencing reactions were performed as indicated above using primers used in amplification or internal primers as indicated in S6 Table. Sequences were aligned to the human genome reference sequence, and breakpoints are depicted in S6 Fig. We had previously reported the sequence across the junction in P255 [24].

Jct1 Analyses

PCR was conducted across Jct1 from DNAs prepared from patients with DUP-TRP/INV-DUP CGRs using a QIAGEN Multiplex PCR. Two control DNAs duplicated through this region and three control DNAs with a single copy at this locus were amplified in parallel. Along with a dystrophin primer pair (Hdys 23F-6FAM and Hdys 23 R) for a single copy region of the human dystrophin gene, we used primers pairs V362H12-F19–6FAM and V362H12-R19 (red arrows), and V362H12-F24–6FAM and V362H12-R24 (black arrows), and V362H12-F19–6FAM and V362H12-R24 (one red, one black arrow) (Figs. 3E, S8, S6 Table). Fluorescently labeled PCR products were diluted 1:100 in sterile HPLC water and subjected to capillary electrophoresis using an ABI PRISM 3130 XL DNA Analyzer. Copy number analysis was performed as previously described using the Peak Scanner software [24].

To subclone breakpoints in PMD DUP-TRP/INV-DUP patients, we amplified patient DNAs containing rearrangements (from BAB1612/P374, BAB2389, and BAB1290) with PCR primers that anneal within the A1a and A1b LCRs and uniquely flanking primers. This yielded four overlapping segments of the two LCRs (S6 Fig). These PCR products were then subjected to electrophoresis in crystal violet 0.8% agarose gels, purified using the SNAP purification kit from Invitrogen, and cloned into TOPO XL cloning vectors. Resultant clones for each of the four segments were screened by digestion and sequenced in their entirety. At least two clones for each region, obtained from independent PCR reactions, were screened for the breakpoint and the corresponding A1a or A1b region. Sequence analysis was conducted using the Lasergene 9 DNA analysis software suite.

Copy Number Analysis of Junctions in the Quadruplication by dPCR

Copy number of junctions in the quadruplication patients and a carrier were determined by dPCR using QuantStudioTM 3D Digital PCR System (Life Technologies), according to the manufacturer’s instructions. Concentration of DNA was determined by QubitR dsDNA BR assay (Life Technologies) using the Qubit 2.0 fluorometer (Life Technologies). Sample DNA was digested with SphI (NEBiolabs) to separate multiple copies of interest that may be located on the same molecule without disrupting the region of amplification. Digests were performed using 400 ng of DNA in a 10 μl reaction containing 10U of SphI and incubating at 37°C for 1.5 hr, followed by heat-inactivation of the enzyme at 65°C for 20 min. The digest was diluted to 40 μl with RNase-free water to yield a concentration of 10 ng/μl DNA. Primers and probes used in the dPCR assays are in S6 Table. Reactions for dPCR included 1x QuantStudioTM 3D Digital PCR Master Mix, 1x TaqMan Copy Number Reference Assay for human RNaseP (Life Technologies, Cat. # 4403328, VIC label), 1–1.5x PrimeTime qPCR 5’ nuclease assay (IDT, FAM label) for jct1 or jct2/3 and 40–60ng DNA in a 16 μl volume. Fifteen μl of this mix was used to load the Digital PCR 20K Chip (Life Technologies); chips were processed according to the manufacturer’s instructions.