Development and validation of a CGH microarray for clinical cytogenetic diagnosis

Abstract

Purpose: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). Methods: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. Results: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%.Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. Conclusion:The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype.Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.Genet Med 2005:7(6):422-432.

Comparison of chromosome analysis and chromosomal microarray analysis: what is the value of chromosome analysis in today’s genomic array era?

“…CMA was performed according to the manufacturers' instructions, with minor modifications as described previously. 8,9 The arrays used evolved from bacterial artificial clone arrays (V3-V6), which were targeted arrays with increased coverage in known disease regions but limited coverage in the backbone, to whole-genome 105K No copy number changes were detected in cases 7-10 using a targeted array. However, the abnormalities were detected using the current wholegenome array (V8 CMA oligonucleotide arrays (V7) 10 and the current version of the whole-genome array (V8), which has ~180,000 oligonucleotides including exon coverage for 1,700 disease or candidate genes.…”

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.…”

“…11,14 -19 We designed a targeted microarray of 366 large genomic clones covering the 41 clinically relevant subtelomeric regions as well as genomic positions corresponding to 55 different genomic disorders, validated its use for molecular cytogenetic diagnosis on samples with known abnormalities, and completed over 1,500 clinical cases on referred blood samples. 11 Based on this collective experience with array-CGH, we carried out a prospective study to assess the feasibility of offering array-CGH in the clinical prenatal diagnostic setting with informed consent and counseling, including reliance on direct analysis without culturing cells. We present here the results of a pilot study on 98 prenatal specimens from current pregnancies to compare results obtained by standard karyotype and array-CGH.…”