Array-Based Comparative Genome Hybridization in Clinical Genetics

Abnormalities in DNA copy number are frequently found in patients with multiple anomaly syndromes and mental retardation. Array-CGH is a high resolution whole-genome technology which improves detection of submicroscopic aberrations underlying these syndromes. Eight patients with mental disability, multiple congenital anomalies and dysmorphic features were screened for submicroscopic chromosomal imbalances using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously detected by FISH analysis were confirmed in two patients, and accurate diagnosis was provided in two previously undiagnosed complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia, cryptorchidsm and hypopigmentation were detected with few discrepancies between the array results and FISH analysis. Contiguous microdeletion of GSCL, HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with aniridia, a borderline false negative WT1 deletion was observed, most probably because of differences between the size of the genomic deletion and the microarray probe. A false positive rate of 0.2% was calculated for clone-by-clone analysis, while the per patient false positive rate was 20%. Array-based CGH is a powerful tool for the rapid and accurate detection of genetic disorders associated with copy number abnormalities, and can significantly improve clinical genetic diagnosis and care. Keywords: comparative genome hybridization (CGH) High-molecular weight genomic DNA was purified from patients' ’peripheral blood leukocytes. Test DNA and normal reference DNA (from an individual of the opposite sex of the test sample, except for N1 and N2) was labeled to incorporate Cy3 or Cy5 fluorophores. Equal aliquots of labeled test and reference DNA were combined and hybridized to GenoSensor Array 300 (GPL3709). This array contains 287 genomic clones, including those for each human telomere, as well as all of the known microdeletion syndromes and additional selected loci representing each chromosome arm. The GenoSensor CGH Array 300 clone list is available at http://www.vysis.com/PDF/GenoSensor300ClonesAndKey_July2004.pdf. Hybridization signal images in three colors, Cy3, Cy5 and DAPI blue were then analyzed by the GenoSensor reader system based on DAPI staining that identified target spots and their location on the grid. By analyzing the set of Cy3/Cy5 ratios (test-to-reference ratios) on all targets, the GenoSensor Array 300 Reader Software (version 1) calculates the ratio most representative of the modal DNA copy number of the sample DNA. For each target the normalized ratio, relative to the modal DNA copy number is calculated. This normalized ratio of the target indicates the degree of gain or loss of copy number, and for each clone the copy number changes are presented. For each target, a P value is calculated automatically by the GenoSensor Array 300 Reader Software (Abbott Vysis), and a P value of 0.005 or less was considered significant.