The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements

Abstract

The constitutional t(11;22)(q23;q11) is the most common recurrent non-Robertsonian translocation in humans. The breakpoint sequences of both chromosomes are characterized by several hundred base pairs of palindromic AT-rich repeats (PATRRs). Similar PATRRs have also been identified at the breakpoints of other nonrecurrent translocations, suggesting that PATRR-mediated chromosomal translocation represents one of the universal pathways for gross chromosomal rearrangement in the human genome. We propose that PATRRs have the potential to form cruciform structures through intrastrand-base pairing in single-stranded DNA, creating a source of genomic instability and leading to translocations. Indeed, de novo examples of the t(11;22) are detected at a high frequency in sperm from normal healthy males. This review synthesizes recent data illustrating a novel paradigm for an apparent spermatogenesis-specific translocation mechanism. This observation has important implications pertaining to the predominantly paternal origin of de novo gross chromosomal rearrangements in humans.

Fig. 1

Palindrome-mediated translocation in humans. (a) Schematic representation of the t(11;22)(q23;q11). The PATRR11 and PATRR22 are located at the breakpoints on 11q23 and 22q11, respectively. (b) FISH analysis of metaphase chromosomes derived from a balanced t(11;22) carrier using a BAC spanning the breakpoint on chromosome 11. The signal from the chromosome 11 BAC is split by the translocation and appears on both the der(11) and the der(22) (left). The same FISH image was inverted to greyscale to show the location of the relevant chromosomes in the metaphase spreads (right). (c) Predicted secondary structure for the palindromic sequence. Short palindromic sequences have the potential to form double-stranded cruciform structures by intrastrand-base pairing in single-stranded DNA. DNA sequences indicated by blue arrows are complementary to those indicated by red arrows. (d) Cruciform extrusion of the plasmid harboring the PATRR11. A plasmid bearing the PATRR11 insert was fixed with psoralen treatment followed by ultraviolet exposure. The PATRR11 fragments were released with restriction enzyme digestion and were visualized using AFM. AFM, atomic force microscopy; BAC, bacterial artifical chromosome; FISH, fluorescent in situ hybridization; PATRR, palindromic AT-rich repeat.

Fig. 2

Polymorphisms of the palindrome affect the de novo translocation frequency. (a) Location of PCR primers. Arrows indicate each arm of the PATRR11 (solid arrows) and PATRR22 (hatched arrows). Size polymorphisms of the PATRR11 were examined by PCR using primers indicated by red and pink triangles. Translocations were detected using one of the primers flanking the PATRR11 (red or pink triangles) and with one of the primers flanking the PATRR22 (blue or green triangles). Centromeres are represented by circles. (b) Strategy for estimation of translocation frequency by PCR. Genomic DNA was extracted from sperm samples. Translocation-specific PCR was performed using multiple batches of template DNA. The translocation frequency was calculated using the equation, q = 1 − (1 − p)^1/n; with n = number of haploid genomes per aliquot, p = the probability that an aliquot contains a translocation, product and q = the probability that one randomly selected haploid genome in a given aliquot sustained a translocation. The gel images show representative PCR results. The upper panel shows results derived from sperm DNA, whereas the lower panel presents results from lymphoblast DNA. Lane M, size marker; lane N, negative control; lane P, genomic DNA from a t(11;22) balanced carrier serving as a positive control. (c) Characterization of the PATRR11 variants. Arrows indicate each unit of inverted repeats. Vertical arrowheads indicate the center of the palindromic sequence. Dotted blue lines show the deleted region, while red lines indicate the insertion of sequences of unknown origin. Other characteristics and the frequency in the general population are shown on the right. (d) A histogram showing the probability of de novo translocation by allele types. Each bar represents the mean value and the vertical bar indicates standard deviation on a log scale. The allele types are abbreviated. PCR, polymerase chain reaction; PATRR, palindromic AT-rich repeat.

Fig. 3

Plasmid-based model system of palindrome-mediated translocation. (a) Schematic representation of the detection system. When the two plasmids harboring PATRR11 and PATRR22 are rearranged within each PATRR region, the transcript from the fusion product splices out the intervening junction sequence and expresses the downstream GFP gene product. (b) Strategy for preparing PATRR plasmids with or without cruciform extrusion. Plasmid purified from Escherichia coli is negatively supercoiled (upper). The PATRR plasmid with negative superhelicity energetically favors cruciform extrusion forming intrastrand-base pairing. The positive free energy of cruciform formation is offset by relaxation of the negative superhelical density (left). If the negative superhelicity is abrogated by incubation with topoisomerase I prior to cruciform extrusion, a relaxed plasmid without the extruded cruciform can be obtained (right). (c) The incidence of translocation-like rearrangements following the use of different preparative techniques for PATRR plasmids. GFP-positive cells were counted after co-transfection with various levels of cruciform-extruding PATRR plasmids. The plasmids were prepared by the alkaline-SDS method (1) and by the Triton method followed by either incubation in various NaCl concentrations (2, 10 mM; 3, 50 mM; 4, 200 mM) or topoisomerase I treatment (5). Representative AFM images of the PATRR plasmid are indicated as follows: arrow indicates cruciform extrusion of the PATRR plasmids. (d) The degree of cruciform in the transfected plasmids affects the levels of rearrangement. The degrees of cruciform extrusion observed by AFM correlate with the ratio of GFP-positive cells (right; r = 0.992, p = 0.0008). AFM, atomic force microscopy; GFP, green fluorescence protein; PATRR, palindromic AT-rich repeat; SDS, sodium dodecyl sulfate.