Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array

Abstract

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Figure 1

Whole genome array-CGH profiles of the MDA-MB-231 using 2 different arrays. (A) Profile by 30k oligoarray and (B) Profile by 3k BAC array. X axis represents individual chromosomes and Y axis represents the signal intensity ratio (tumor/normal) in log₂ ratio. Red dots represent the probes above ratio zero and green dots represent below zero.

Figure 2

Validation of the 17p12 copy number gain identified by oligoarray-CGH. (A) FISH analysis of the MDA-MB-231. The signal number of 17p12 (red arrow) is higher than the signal number of 2q35 where no CNA was identified by oligoarray-CGH (green arrow). (B) Genomic qPCR analysis of MDA-MB-231. The signal intensity ratio of the test DNA (MDA-MB-231) is 1.9 (SD = 0.09).

Figure 3

Genomic qPCR results of 7 CNA regions identified by the oligoarray only (gains on 1q23.3, 16q23.1, 18q21.1 and 19q13.43, and losses on 6p12.3, 10q26.13 and 22q13.31), and a non-CNA region (2q35).

Figure 4

Integrated analysis of copy number and expression profiles. (A) Correlation analysis of copy number status and expression levels. X axis represents the array-CGH signal intensity ratio (tumor/normal) in log₂ scale and Y axis represents the expression signal intensity ratio (tumor/normal) in log₂ scale. Tumor, MDA-MB-231; Normal, normal female genomic DNA (B) Example of the correlation at an amplified region (17p12-p11.2). The arrow indicates the highest value of expression signal intensity ratio (7.6 in log₂ scale, PMP22 gene). (C) Example of the correlation at a deleted region (9p22.1-p21.2). The arrow indicates the lowest value of expression signal intensity ratio (-6.8 in log₂ scale, CDKN2AB gene). In B and C, upper box represents whole chromosome arm plot and lower box represents copy number-expression signal intensity ratios in the region selected from the upper box. Both DNA copy number signal intensity ratio (solid bar) and RNA expression signal intensity ratio (gray bar) of each probe are represented in log₂ scale. X axis represents the chromosomal position in mb scale and Y axis represents the signal intensity ratio in log₂ scale.