Chromosome copy number analysis in screening for prognosis-related genomic regions in colorectal carcinoma

Abstract

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism-typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large-scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis-related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development.

Figure 1

Chromosomal copy number alterations and loss of heterozygosity (LOH) in the colorectal carcinoma genome. (a) The study subjects (n = 94) were subjected to a hierarchical clustering analysis based on the inferred copy number for all autosomal single nucleotide polymorphism (SNP) sites. Copy number is color coded according to the indicated scheme at the bottom. SNP sites are ordered by their physical position from top to bottom, and the borders between chromosomes are indicated by small bars at the left. (b) Chromosome copy number (left panel) and LOH likelihood score (right panel) are demonstrated for patient ID#002 in a chromosome view in a symmetrical manner. Copy number value is color coded as in (a), and LOH likelihood score is colored according to the scheme indicated at the bottom. Chromosome numbers are shown at the center. The allele‐specific copy number data for the 3p region (indicated by a blue circle) is demonstrated in the inset as pink and green lines. Below the cytoband figure, the positions of SNP sites with a hetero‐ or discordant‐call are indicated by green or pink bars, respectively.

Figure 2

Verification of copy number changes. (a) The DNA quantities of PER3, IIP34, FAT, and BRCC2 (relative to that of GAPDH) were measured by real‐time polymerase chain reaction in the subjects with inferred copy number two (n = 2) or one (n = 1). The mean + SD value for each gene was normalized to the corresponding mean value for the group with diploid chromosomes, and is shown in the left panel. The mRNA amount for each gene (relative to that of GAPDH) was also quantitated by real‐time reverse transcription–polymerase chain reaction and is shown in a similar way. (b) The relative DNA (left panel) or mRNA (right panel) of MAFK and PTPN1 was calculated as in (a).

Figure 3

Prognosis‐related copy number (CN) loss and loss of heterozygosity (LOH). The survival of the subjects with or without copy number loss of a locus at (a) chromosome 5 or (b) chromosome 16 was compared using Kaplan–Meier analysis. The P‐value for each comparison was calculated using the log rank test.