Use of targeted array‐based CGH for the clinical diagnosis of chromosomal imbalance: Is less more?

Abstract

Chromosome analysis is an important component to the diagnosis of congenital anomalies, developmental delay, and mental retardation. Routine chromosome analysis identifies aneuploidy and structural rearrangements greater than 5 Mb but cannot identify abnormalities of the telomeric regions or microdeletions reliably. Molecular cytogenetic techniques were developed to overcome these limitations. High-resolution comparative genomic hybridization (CGH)-based microarrays (array CGH) were developed to increase the resolution of chromosomal studies and to provide a comprehensive assay by using large-insert clones as the target for analysis. We constructed a microarray for the clinical diagnosis of medically significant and relatively common chromosomal alterations. Nine hundred six bacterial artificial chromosome (BAC) clones were chosen, the chromosomal locations of which were confirmed by fluorescence in situ hybridization (FISH). FISH-testing showed that 7% of the clones were mismapped based on map locations obtained from two publicly available databases (58 mapped to the wrong chromosome and three mapped to a different locus on the same chromosome), 16% cross-hybridized to other chromosomes, and 12% did not hybridize or showed poor hybridization signals under uniform FISH conditions. Thus, from a total of 906 BAC clones that were evaluated, only 589 (65%) were deemed adequate for arraying on this clinical device. The performance of this array was tested in a set of blinded experiments on a cohort of phenotypically normal individuals and on individuals with known chromosome abnormalities. The array identified deletion/duplication polymorphisms not seen by FISH in the phenotypically normal individuals and detected single copy dosage differences in all of the cases with known chromosomal abnormalities. All abnormalities detected by the array were confirmed by FISH with BACs from the appropriate loci. Our data demonstrate that the rigorous assessment of BACs and their use in array CGH is especially important when the microarray is used for clinical diagnosis. In addition, this study illustrates that when constructed carefully with proper attention to the quality of the BACs that are arrayed, array CGH is an effective and efficient tool for delineating chromosomal aberrations and an important adjunct to FISH and conventional cytogenetics.

Simple detection of genomic microdeletions and microduplications using QMPSF in patients with idiopathic mental retardation

“…17 -19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories. 35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements. 36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR. Our present experience with other applications of these methods indicates that both provide similar sensitivity of detection.…”

Impact of genotype-first diagnosis: the detection of microdeletion and microduplication syndromes with cancer predisposition by aCGH

“…16 The current version of the SignatureChip, the SignatureChip Whole Genome ® (SignatureChipWG), contains 4670 BACs representing 1543 loci with each locus represented by a minimum of three overlapping clones. The subtelomeric and pericentromeric regions are represented with a higher density of overlapping BAC clones, targeted to the unique sequences adjacent to these repetitive regions and consisting of contigs of clones located approximately every 0.5 Mb spanning Ͼ5 Mb.…”

NF1 microduplications: identification of seven nonrelated individuals provides further characterization of the phenotype

“…Targeted bacterial artificial chromosome-based microarray analysis was originally performed on DNA from subjects 1, 2, and 3 as previously described. 24 Whole-genome bacterial artificial chromosome-based microarray analysis was originally performed on DNA from subject 4 as previously described. 17 Oligonucleotide-based microarray analysis was originally performed on DNA from subjects 5, 6, and 7 using a custom 12-plex 135K-feature whole-genome oligonucleotide microarray (SignatureChip Oligo Solution v2.0, custom-designed by Signature Genomic Laboratories, Spokane, WA, and manufactured by Roche NimbleGen, Madison, WI) using previously described methods.…”