Background: Sporadic colorectal tumors probably carry genetic alterations that may be related to familiar clusters according to risk loci visualized by SNP arrays on normal tissues. The aim of the present study was therefore to search for DNA regions (copy number variations, CNVs) as biomarkers associated to genetic susceptibility for early risk predictions of colorectal cancer. Such sequence alterations could provide additional information on phenotypic grouping of patients. Material and Methods: High resolution 105K oligonucleotide microarrays were used in search for CNV loci in DNA from tumor-free colon mucosa at primary operations for colon cancer in 60 unselected patients in comparison to DNA in buffy coat cells from 44 confirmed tumor-free and healthy blood donors. Array-detected CNVs were confirmed by Multiplex ligation-dependent probe amplification (MLPA). Results: A total number of 205 potential CNVs were present in DNA from colon mucosa. 184 (90%) of the 205 potential CNVs had been identified earlier in mucosa DNA from healthy individuals as reported to the Database of Genomic Variants. Remaining 21 (10%) CNVs were potentially novel sites. Two CNVs (3q23 and 10q21.1) were significantly related to colon cancer, but not confirmed in buffy coat DNA from the cancer patients. Conclusion: Our study reveals two CNVs that indicate increased risk for colon cancer; These DNA alterations may have been acquired by colon stem cells with subsequent appearance among epithelial mucosa cells. Impact: Certain mucosa CNV alterations may indicate individual susceptibility for malignant transformation in relationship to intestinal toxins and bacterial growth.
Confirmed large-scale copy number DNA variations were observed in our previous analyses on colon mucosa tissue from colorectal cancer (CRC) patients with different clinical outcome [
DNA sequence alterations such as CNVs in mucosa tissue from patients with colorectal cancer may either be acquired and reflect toxic environments to which colon epithelial cells are exposed during life time predisposing to carcinogenesis, or it may represent inborn host susceptibility for cancer development [
A total cohort of 486 patients diagnosed with primary colorectal carcinoma were subgrouped into Dukes A, B, C and D tumor stages corresponding to stage I-IV according to histopathological findings at operation [
Tissue biopsies were homogenized and total genomic DNA extraction was performed with QIamp DNA mini kit (Qiagen, Hilden, Germany) according to instructions and quantified in a NanoDrop ND-1000 (NanoDrop Technologies Inc., USA). The buffy coat layer from blood was used for genomic leukocyte DNA extraction with Flexi Gene DNA kit (Qiagen, Hilden, Germany).
DNA was hybridized in competition with the commonly used reference DNA NA10851 (Coriell Cell Repositories, Camden, NJ, USA) to 105K Whole Human Genome oligo arrays (Design 014698, Agilent Technologies, Palo Alto, CA, USA). The high resolution 105K array contains approximately 99000 coding and noncoding human sequences distributed with median 21.7 kb probe spacing genome wide. Genomic DNA derived from colon mucosa tissue in 5 patients was pooled into one analytical specimen repeated to give triplicates for all Dukes A-D groups (n = 12 arrays). Genomic DNA from 5 individual blood samples from Dukes A patients was also pooled to one analytical specimen (n = 1 array). Thus, 12 analytical specimens of pooled DNA from colon mucosa and 1 specimen of pooled DNA from patients’ blood cells were analyzed versus reference DNA. 500 ng of study and reference DNA were labeled with Agilent Genomic DNA Labeling Kit PLUS, hybridized with Agilent Oligo array CGH Hybridization Kit and washed with Agilent Oligo Wash Buffer 1 & 2 set, following Agilents standard processing recommendations. All labeled samples were checked in a NanoDrop prior to hybridization and arrays were scanned on an Agilent scanner (G2565AA, Agilent Technologies). Analyses of scanned images from two-color oligonucleotide CGH arrays were performed in Feature Extraction 9.5 (FE 9.5, Agilent Technologies), using default CGH settings. FE 9.5 result files were implemented in CGH Analytics 3.4 and quality control was assessed by metrics provided by the software.
MLPA analysis was performed to confirm array CGH CNVs using a probe mixture (Salsa MLPA kit P300-A1 Human DNA Reference-2, MRC-Holland b.v., Amsterdam, the Netherlands) with 20 different probes and 11 specific probes. MLPA is an effective approach to cover several gene sequences in single DNA samples with confirmed quantitative information compared to results achieved by array CGH. The specific probes were designed to study specific known CNVs and were distributed on different chromosomes shown in
The MLPA analysis was performed according to the protocol provided by the supplier with minor changes; denaturation of DNA was prolonged to 10 min. Briefly, 250 - 500 ng DNA in 5 μl TE was denatured at 98˚C and subsequently hybridized overnight (16 hours) with a mix of probes, each consisting of two parts that recognize adjacent target sequences. After hybridization, the probes were ligated with a thermostable ligase. PCR was performed on ligated products with two universal PCR primers, amplifying all probe pair in one reaction. 2 μl of the PCR products were diluted with 9 μl High Dye Formamide and 0.3 μl size standard LIZ (Applied Biosystems, Foster City, CA, USA). The amplification products were separated by electrophoresis using an ABI 3730 Genetic Analyzer (Applied Biosystems). The MLPA results were analyzed by GeneMapper software (v. 3.7, Applied Biosystems) and the values were normalized against a normal genome.
Standard DNA reference sample, NA10851 was used in comparison to study specimens. A study specimen contained pooled DNA from 5 patients in CGH analysis. Impact of possible reference specific CNVs was reduced by hybridization of the NA10851 reference DNA to another purchased reference sample, which is a pool of DNA from normal colon mucosa derived from six human cancer free donors (Biochain Institute Inc., Hayward, CA, USA). This procedure was used to attenuate false positive calls.
For CGH analyses the ADM-2 algorithm was used to determine statistically significant sequence alterations for identification of potential CNV intervals. Data were centralized and calls with average log2 ratios < 0.3 were excluded from analysis [
Chromosome | Start | Stop | N◦ of arrays | N◦ of probes | Genes |
---|---|---|---|---|---|
1p35.3 | 28884507 | 28884907 | 2 | 1 | Y |
1p13.3 | 109944151 | 109944551 | 5 | 1 | N |
1q32.1 | 202959453 | 202959853 | 4 | 1 | N |
10p12.1 | 29111554 | 29111954 | 2 | 2 | N |
10q22.1 | 72792179 | 72792579 | 2 | 1 | Y |
11q13.2 | 68249211 | 68249611 | 3 | 1 | Y |
11q24.2 | 125820710 | 125821110 | 3 | 6 | Y |
12q13.11 | 45052367 | 45052767 | 2 | 1 | Y |
17q24.3 | 65049247 | 65049647 | 2 | 3 | Y |
2p24.3 | 14723477 | 14723877 | 3 | 1 | Y |
2p23.3 | 25040673 | 25041073 | 3 | 1 | Y |
2p22.2 | 38176230 | 38176630 | 4 | 1 | N |
21q22.13 | 38210261 | 38210661 | 2 | 1 | Y |
22q12.3 | 31703891 | 31704291 | 9 | 1 | Y |
4p11 | 49422536 | 49422936 | 2 | 1 | N |
4q21.3 | 88154362 | 88154762 | 2 | 1 | Y |
4q35.1 | 185282603 | 185314025 | 2 | 3 | Y |
5q14.1 | 76969795 | 77002969 | 2 | 5 | Y |
5q31.1 | 133734640 | 133735040 | 3 | 1 | Y |
6q21 | 109714925 | 109715325 | 5 | 1 | N |
6q22.31 | 123422159 | 123422559 | 2 | 1 | Y |
Y = yes; N = no.
alteration according to defined confidence limits. Each call was verified by comparison of altered locus to all known variations in the Database of Genomic Variants (DGV, Build 36 (Mar. 2006), last update 3 Jan 2014
(http://projects.tcag.ca/variation/) [
Genome-wide analyses of sequence variations in all patient groups defined by Dukes tumor stages, revealed a total number of 774 potential CNV loci calls identified in DNA from colon mucosa in 60 patients when compared to a reference DNA (NA10851). 205 calls were identified in at least two specimens, where 90% (185) were present in 2 - 6 specimens and 10% [
Identified CNVs were present in more than 50% of the mucosa specimens in either early (Dukes A and B, n = 6) or late (Dukes C and D, n = 6) tumors. No significant correlation between mucosa CNV and tumor stage/pro- gression was confirmed in our data set. However, three mucosa CNVs discriminated between early (Dukes A and B) and late (Dukes C and D) stages. One multi probe variation at 15q21.3 was present in early Dukes stages (2 Dukes A and 1 Dukes B specimen), and one single probe variation at 2p15 was present in early Dukes stages (2 Dukes A and 1 Dukes B). One single probe variation, identified at 10q21.1, was present in late Dukes stages (2 Dukes C and 1 Dukes D,
Selected mucosa CNV loci from array CGH were confirmed and compared to DNA from healthy blood donors with Multiplex ligation-dependent probe amplification (MLPA) analyses on an individual patient basis. Of 11 mucosa locus that were significantly changed with array CGH, two (3q23 and 10q21.1) were significantly more frequent in colorectal cancer patients compared to buffy coat DNA from healthy blood donors (p < 0.001). None of these mucosa CNVs were detected in blood from Dukes A cancer patients (
Colorectal cancer is divided into hereditary (~20%) and sporadic (~80%) forms. Among patients with sporadic disease, some may carry familiar risk genotypes [
Potential CNV Sites* | Locus | Start Position | Probe Size | Genes | Reported Elsewhere |
---|---|---|---|---|---|
CNV in DNA specimens from patients with Dukes A-D tumors | |||||
3q23 | 142603243 | Single | ZBTB38 | ||
5q21.1b | 100841957 | Single | - | ||
7q36.1a | 151894081 | Single | - | ||
9q32a,b | 113501980 | Single | c9orf84 | ||
13q31.2 | 86720225 | Single | - | ||
15q24.1 | 71951164 | Single | - | ||
15q25.1a | 76715721 | Single | CHRNB4 | Lung cancerc | |
22q12.3d | 31703891 | Single | SYN3 | ||
CNV in patients with early tumors (Dukes A and B) | |||||
2p15b | 63189402 | Single | - | ||
15q21.3b | 54911220 | Multi (14,988 kb) | - | ||
CNV in patients with late tumors (Dukes C and D) | |||||
10q21.1a | 54188864 | Single | - |
*CNVs were regarded potential if present in more than 50% of the specimens and unrecognized in colorectal cancer at that time. aColocalized with CNV sites detected by de Smith et al. [
Probe locus | Alteration | Array CGH Altered CRC Samples (n = 12) | MLPA Altered CRC Samples (n = 39) | MLPA Altered Control Samples (n = 41) |
---|---|---|---|---|
2p15 | Deletion | 3 (25%) | 1 (3%) | 6 (15%) |
3q23 | Deletion | 6 (50%) | 13 (38%)* | 1 (2%) |
5q21.1 | Amplification | 9 (75%) | 2 (6%) | 2 (5%) |
7q36.1 | Deletion | 9 (75%) | 0 | 0 |
9q32 | Amplification | 7 (58%) | 2 (6%) | 2 (5%) |
10q21.1 | Deletion | 3 (25%) | 12 (35%)* | 4 (10%) |
13q31.2 | Amplification | 9 (75%) | 3 (9%) | 8 (20%) |
15q21.3 | Amplification | 3 (25%) | 0 | 5 (12%) |
15q24.1 | Deletion | 6 (50%) | 5 (15%) | 0 |
15q25.1 | Deletion | 10 (83%) | 3 (9%) | 3 (7%) |
22q12.3 | Amplification | 9 (75%) | 2 (6%) | 5 (12%) |
*p < 0.0001 with Bonferroni corrected Fischer’s exact test versus healthy donors DNA. An array CGH sample contained pooled DNA from mucosa tissue from 5 patients.
our previous report [
Our data revealed 184 known and 21 novel CNVs present in pooled DNA from array CGH based analysis in comparisons with the 109,863 CNV loci present in the Database of Genomic Variants (DGV). None of the 21 novel CNVs was discovered in all mucosa specimens, but one CNV (22q12.3) was found in more than 75% of the mucosa specimens indicating that identified potential CNVs are either loci in DNA from Northern Europeans or may be sequences related to risk for colorectal cancer. Most of the variants were detected by one single probe. Out of the 21 novel CNV intervals identified in our data set, 15 (71%) contained at least one gene. With few exceptions, these genes did not code for any known tumor related gene, which is in line with results from the majority of the GWAS performed to date [
From array CGH analyses we found three potential CNV regions (10q21.1, 15q21.3, 2p15) that may discriminate early (Dukes A and B) from late (Dukes C and D) tumor stage, present in 25% - 30% of all analyzed mucosa specimens. One of these CNVs, located at 10q21.1 was only found in early tumor stages based on MLPA analyses at variance with other observations [
During last years, several genome-wide association studies (GWAS) have presented a number of potential and valid risk sites for each of four prevalent cancer types, as breast, prostate, colorectal and lung cancer [
In conclusion, oligonucleotide-based array CGH appears as a sensitive tool for screening for identification of CNVs related to tumor development, complementary to the use of SNP arrays. Two CNVs at 3q23 and 10q21.1 were identified and statistically validated on an individual basis to discover significant risk for colorectal cancer. Our findings need further validation in large patient cohort studies.
Supported in parts by grants from the Swedish Cancer Society (CAN 2010/255), the Swedish Research Council (08712), Tore Nilson Foundation, Assar Gabrielsson Foundation (AB Volvo), Jubileumskliniken Foundation, IngaBritt & Arne Lundberg Research Foundation, Swedish and Gothenburg Medical Societies and the Medical Faculty, University of Gothenburg, VGR 19/00, 1019/00.