Large inverted repeats within Xp11.2 are present at the breakpoints of isodicentric X chromosomes in Turner syndrome

Abstract

Turner syndrome (TS) results from whole or partial monosomy X and is mediated by haploinsufficiency of genes that normally escape X-inactivation. Although a 45,X karyotype is observed in half of all TS cases, the most frequent variant TS karyotype includes the isodicentric X chromosome alone [46,X,idic(X)(p11)] or as a mosaic [46,X,idic(X)(p11)/45,X]. Given the mechanism of idic(X)(p11) rearrangement is poorly understood and breakpoint sequence information is unknown, this study sought to investigate the molecular mechanism of idic(X)(p11) formation by determining their precise breakpoint intervals. Karyotype analysis and fluorescence in situ hybridization mapping of eight idic(X)(p11) cell lines and three unbalanced Xp11.2 translocation lines identified the majority of breakpoints within a 5 Mb region, from approximately 53 to 58 Mb, in Xp11.1-p11.22, clustering into four regions. To further refine the breakpoints, a high-resolution oligonucleotide microarray (average of approximately 350 bp) was designed and array-based comparative genomic hybridization (aCGH) was performed on all 11 idic(X)(p11) and Xp11.2 translocation lines. aCGH analyses identified all breakpoint regions, including an idic(X)(p11) line with two potential breakpoints, one breakpoint shared between two idic(X)(p11) lines and two Xp translocations that shared breakpoints with idic(X)(p11) lines. Four of the breakpoint regions included large inverted repeats composed of repetitive gene clusters and segmental duplications, which corresponded to regions of copy-number variation. These data indicate that the rearrangement sites on Xp11.2 that lead to isodicentric chromosome formation and translocations are probably not random and suggest that the complex repetitive architecture of this region predisposes it to rearrangements, some of which are recurrent.

Figure 1.

FISH analysis of idic(X)(p11) cell lines using CEPX probe. Interphase nuclei of (A) GM00088, (B) GM00339, (C) GM00735, (D) GM02595, (E) GM03543, (F) GM08944, (G) GM13166 and (H) metaphase spread of GM13019. idic(X)(p11) chromosomes are noted with red arrows.

Figure 2.

Representative BAC FISH of idic(X)(p11) and translocation cell lines. Shown are FISH images for idic(X)(p11) cell lines: (A) GM00088, (B) GM00339, (C) GM00735, (D) GM02595, (E) GM03543 and (F) GM13019; and translocation line: (G) GM10204. The normal X, idic(X)(p11) and derivative chromosomes are indicated with white, red, and yellow arrows, respectively. Note the supernumerary idic(X)(p11) in GM00339 cells consistent with the identified mosaic karyotype.

Figure 3.

Representative aCGH data of idic(X)(p11) and translocation cell lines. Shown are images for idic(X)(p11) cell lines: GM02595, GM00088 and GM13019; and translocation line: GM05396. Identified breakpoints are indicated with black arrows. The breakpoint for GM02595 occurred within the pericentromeric region, and GM00088 and GM013019 had one and two distinct breakpoints, respectively. Green, black and red dots represent oligonucleotide aCGH probes with log₂ ratios less than −0.25, in between −0.25 and 0.25 and >0.25, respectively.

Figure 4.

Illustration of X chromosome ideogram with enlarged views from ∼37 to 39 Mb and ∼51 to 59 Mb. The location of known human genes from the NCBI reference sequence collection (RefSeq), structural variants (CNV, insertions/deletions, and inversions) from the Database of Genomic Variants (DGV), segmental duplications and oligonucleotide probes used for aCGH are highlighted in blue, orange, green and black, respectively. In addition, the locations of breakpoint intervals identified by aCGH are noted in red.

Figure 5.

Illustration of derivative X chromosome breakpoint intervals and local genomic architecture. The location of known human genes from the NCBI reference sequence collection (RefSeq) are illustrated in blue; CNV and inversions from the DGV are illustrated in orange and red, respectively; segmental duplications are noted and color is used to distinguish level of similarity (gray: 90–98% similarity; yellow: 98–99% similarity; orange: >99% similarity. Paired inverted repeats associated with breakpoints that were identified by the Inverted Repeat Finder algorithm (27) are highlighted with green boxes. Oligonucleotide probes used for aCGH are black and inter-probe breakpoint regions are noted in red. In addition, some regions display low complexity repeats identified by RepeatMasker (http://www.repeatmasker.org/). These include short interspersed elements (SINEs), long interspersed nuclear elements (LINEs) and long terminal repeat elements (LTRs) and the lighter shading indicates increased base mismatch, deletion and insertion associated with a repeat element. Breakpoint regions are labeled ‘A’ through ‘I’ in accord with the panels in Figure 6.

Figure 6.

Dot matrix plots for each breakpoint region showing regions of similarity based upon alignment using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default algorithm parameters. (A)–(I) correspond to breakpoint regions labeled ‘A’ to ‘I’ in Figure 5. In the selected cases that included adjacent flanking sequence, the identified breakpoint regions are highlighted with red boxes. Both the x- and y-axes represent nucleotide sequence (in kilobases) from each breakpoint region. Alignments are shown in the plots as lines, whereby directly orientated homologies are slanted from the bottom left to the upper right corner, and inverted homologies are slanted from the upper left to the lower right.