Development of a comparative genomic hybridization microarray and demonstration of its utility with 25 well-characterized 1p36 deletions

Abstract

Chromosomal abnormalities, such as deletions and duplications, are characterized by specific and often complex phenotypes resulting from an imbalance in normal gene dosage. However, routine chromosome banding is not sensitive enough to detect subtle chromosome aberrations (<5-10 Mb). Array-based comparative genomic hybridization (array CGH) is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by co-hybridizing differentially labeled test and control DNAs to a microarray of genomic clones. We used a previously assembled contig of large-insert clones that span 10.5 Mb of the most distal region of 1p36 to design a microarray. The array includes 97 clones from 1p36, 41 clones from the subtelomeric regions of all human chromosomes, and three clones from each of the X and Y chromosomes. We used this microarray to study 25 subjects with well-characterized deletions of 1p36. All array CGH results agree with the deletion sizes and locations of the breakpoints in these subjects as determined previously by FISH and microsatellite analyses. Terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements were also identified. We anticipate that array CGH will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization

“…11,20 For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples, followed by integration of the data from both dye-reversed hybridizations to determine inferences for each case (Fig. 1).…”

“…The quantitation data were subjected to normalization as described previously, and the dye-reversed data were combined to determine a single fold-change value for each clone. 11,20 Inferences were made for all clones using these final combined data values (Fig. 1).…”

“…1). 11,20 All analyses were performed on log 2 ratios using code for the normalization and inference that was implemented in the R statistical programming language.…”

Delineation of mechanisms and regions of dosage imbalance in complex rearrangements of 1p36 leads to a putative gene for regulation of cranial suture closure

“…The construction of the microarray 20 and BAC/PAC clones used 12 have been published. Genomic DNA was isolated from lymphoblastoid cell lines established from four subjects (64,69,71,85) with known 1p36 rearrangements and from peripheral blood of a phenotypically normal male reference.…”

“…19 We used a dye-reversal strategy on two separate microarrays in which study subject and reference DNAs were labeled with cyanine 3 (Cy3) and cyanine 5 (Cy5), respectively, cohybridized to one microarray and then oppositely labeled and cohybridized to a second microarray. 20,21 Images were acquired using a GenePix 4000B (Axon Instruments) dual-laser scanner in combination with GenePix Pro 4.0 imaging software. Two simultaneous scans of each array were obtained at wavelengths of 635 and 532 nm, and data analysis was performed as described previously 20 to identify gains or losses within this particular region of 1p36.…”