High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization

Abstract

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.

Figure 1

Telomere screening by array-based CGH. A–C, Telomere profiles of three patients. The arrays were composed of 77 cloned subtelomeric genomic DNA targets, ordered within each chromosome from pter to qter. Results for clone GS-963-K6, located on 4qter, are not included because of the high variability observed in the normal-versus-normal hybridizations (see the “Material and Methods” section). The unblackened squares represent the T/R values of the control hybridizations, individually normalized to a value of 1. The vertical lines represent twice the SD for each target clone in the control hybridizations. Next to that, the dark horizontal lines indicate the thresholds for copy-number loss (0.8) and gain (1.2). The blackened squares represent the normalized T/R ratios for patient 2 (46,XY,del[18][q21.2]) (A), patient 8 (46,XY,der[13;14][q10;q10],dup[17][qterq24.2]) (fig. 1B) and patient 14 (46,XX,der[9]t [9;?)[p24;?]) (fig. 1C) versus reference hybridization. A shows a clear loss of both telomeric targets mapping to the subtelomeric region of the long arm of chromosome 18. In B, gain of one 17qter clone and deletion of the other 17qter clone is observed in patient 8. In C, gain of the 7pter clone and loss of both 9pter clones is observed. D, Quadruplicate array-based CGH experiment of patient 10 (46,XY,del[7][q36]). The unblackened squares represent the mean T/R values obtained with the DOP-PCR products, and the blackened squares represent the mean T/R values obtained with the whole clone DNAs as targets. The vertical lines represent the SD for each target clone in the four hybridizations. The deletion of the distal part of the long arm of chromosome 7 is clearly identified in the whole clone DNAs and, to a lesser extent, in the DOP-PCR products (in one experiment, the T/R value for this clone was 0.87). In addition, the SDs of the DOP-PCR products of two clones, GS-1011-O17 on 2qter and GS-546-C11 on 19pter, crossed the threshold for copy-number gain because of a false-positive result in one of the four repeat experiments. E and F, FISH validation experiments of patient 8. The duplicated 17qter clone is labeled in green; the deleted 17qter clone is labeled in red. The duplication is visible in interphase (F), and the deletion is visible in both interphase (F) and metaphase (E). G and H, FISH validation experiments of patient 14 (G metaphase, H interphase). The 7pter clone is labeled in green, and chromosome 7 centromere is labeled in red for chromosome identification. The 7pter clone is present on both chromosome 7 homologs, as well as on the short arm of chromosome 9, thus confirming the unbalanced translocation as identified by array-based CGH.