Comparative analysis of copy number detection by whole-genome BAC and oligonucleotide array CGH

Abstract

Background: Microarray-based comparative genomic hybridization (aCGH) is a powerful diagnostic tool for the detection of DNA copy number gains and losses associated with chromosome abnormalities, many of which are below the resolution of conventional chromosome analysis. It has been presumed that whole-genome oligonucleotide (oligo) arrays identify more clinically significant copy-number abnormalities than whole-genome bacterial artificial chromosome (BAC) arrays, yet this has not been systematically studied in a clinical diagnostic setting.

Results: To determine the difference in detection rate between similarly designed BAC and oligo arrays, we developed whole-genome BAC and oligonucleotide microarrays and validated them in a side-by-side comparison of 466 consecutive clinical specimens submitted to our laboratory for aCGH. Of the 466 cases studied, 67 (14.3%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome BAC array, and 73 (15.6%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome oligo array. However, because both platforms identified copy number variants of unclear clinical significance, we designed a systematic method for the interpretation of copy number alterations and tested an additional 3,443 cases by BAC array and 3,096 cases by oligo array. Of those cases tested on the BAC array, 17.6% were found to have a copy-number abnormality of potential clinical significance, whereas the detection rate increased to 22.5% for the cases tested by oligo array. In addition, we validated the oligo array for detection of mosaicism and found that it could routinely detect mosaicism at levels of 30% and greater.

Conclusions: Although BAC arrays have faster turnaround times, the increased detection rate of oligo arrays makes them attractive for clinical cytogenetic testing.

Figure 1

Identification by oligonucleotide microarray of additional complexity missed by BAC microarray. (A) BAC microarray results showing a single-copy loss of 34 BAC clones from the terminus of 5p, approximately 6.8 Mb in size (chr5: 387,034-7,150,950, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the p-arm probes to the left and q-arm probes to the right. (B) shows oligonucleotide microarray results of the terminal deletion shown in (A) in addition to single-copy gain of 29 probes from 5p, approximately 1.38 Mb in size (chr5: 8,511,592-9,888,817, based on UCSC 2006 hg 18 assembly). Probes are ordered as in the BAC array. Regions shaded in blue represent deletions detected by microarray, whereas duplications are shaded in pink.

Figure 2

Oligonucleotide microarray analysis of a mosaic 16q12.1 deletion (shaded blue region). The zoomed-in microarray plot shows a single-copy loss of 289 probes from 16q12.1, approximately 2.77 Mb in size (chr16: 46,837,260-49,605,054, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most proximal 16q11.2 probes to the left and the most distal 16q12.2 probes to the right.

Figure 3

Oligonucleotide microarray analysis of artificially derived mosaic trisomy 21 samples. (A) 10% trisomy 21 showing a very subtle copy-number gain for all clones on chromosome 21. The profile was generated using DNA extracted from a mixture of blood which contained 10% WBCs from a trisomy 21 subject and 90% WBCs from a normal male individual. (B) 15% trisomy 21, generated as in (A), showing a very subtle copy-number gain for all clones on chromosome 21. (C) 20% trisomy 21, generated as in (A) showing a subtle copy-number gain for all clones on chromosome 21. (D) 30% trisomy 21, generated as in (A), showing a clear copy-number gain for all clones on chromosome 21. The inset images to the right of each array plot show the average log₂ ratio of all probes mapping to chromosome 21, with the horizontal dotted line representing a log₂ ratio of zero and the vertical dotted line representing the centromere. A pink bar plotted above the horizontal line represents a copy-number gain of all probes on chromosome 21. To the left of each inset image is the average log₂ ratio at the specified proportion of trisomic cells.

Figure 4

Oligonucleotide microarray characterization of an interstitial deletion at 17p13.3. The zoomed-in microarray plot shows a single-copy loss of six probes from the short arm of chromosome 17 at 17p13.3, approximately 44.0 kb in size (chr17: 2,415,074-2,459,051, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most distal 17p13.3 probes to the left and the most proximal 17p13.3 probes to the right. Below is a schematic of the deletion region. The deletion disrupts the PAFAH1B1/LIS1 gene.

Figure 5

Oligonucleotide microarray characterization of an interstitial deletion at 6q14.1. The zoomed-in microarray plot shows a single-copy loss of 43 oligonucleotide probes from the long arm of chromosome 6 at 6q14.1, approximately 2.9 Mb in size (chr6: 79,838,518-82,730,466, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with the most proximal 6q14.1 probes to the left and the most distal 6q14.1 probes to the right. Below is a schematic of the deletion region. Blue and gray boxes represent genes in the deletion region.

Figure 6

BAC microarray characterization of a 9q33.1 deletion. The zoomed-in microarray plot shows a single-copy loss of three BAC clones from the long arm of chromosome 9 at 9q33.1, approximately 262 kb in size (chr9: 119,452,279-119,714,054 based on UCSC 2006 hg 18 assembly). The nearest distal clone on chromosome 9 that is not deleted is RP11-977E8 and is approximately 4.0 Mb away from the deleted region. The nearest proximal clone on chromosome 9 that is not deleted is RP11-999I23 and is approximately 4.4 Mb away from the deleted region. Probes are ordered on the x axis according to physical mapping positions, with proximal 9q32 clones to the left and distal 9q33.2 clones to the right. Below is a schematic of the deletion region. Vertical blue lines represent the minimum size of this alteration, which encompasses one gene, TLR4.

Figure 7

Oligonucleotide microarray characterization of an interstitial deletion at 4q25. The zoomed-in microarray plots shows a single-copy loss of 15 oligonucleotide probes from the long arm of chromosome 4 at 4q25, approximately 159.6 kb in size (chr4: 108,834,399-108,994,048, based on UCSC 2006 hg 18 assembly). Probes are ordered on the x axis according to physical mapping positions, with proximal 4q25 clones to the left and distal 4q25 clones to the right. Below is a schematic of the deletion region. The deletion disrupts the PAPSS1 and SGMS2 genes, represented by blue boxes.