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Chromosome 8 BAC Array CGH and Expression Studies Identify Amplification and Overexpression of TRMT12 in Breast Cancer

ABSTRACT: Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. Fine mapping of such genomic changes, and evaluation of associated expression changes, are expected to provide reagents for diagnosis, insights into understanding the disease, and open up avenues for novel therapeutic intervention. We made an effort to search for genes on chromosome 8 with altered copy number and expression. A high resolution (0.1Mb) bacterial artificial chromosome (BAC) array of chromosome 8 that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC DNA. Hybridization of DNA from a breast cancer cell line (SKBR3) with two known amplified regions (8q21 and 8q24) resulted in resolving this region into 6 distinct amplicons, in addition identified 3 deleted regions. The boundaries and the peak of each region were verified by FISH, and the extent of amplification/deletion for each region was validated by qPCR. Using these BAC arrays, CGH was performed with a total of eight breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. Two consensus regions in 8q24 included a 1.2 Mb region (125.3-126.5 Mb) and a 1.0 Mb region (128.1-129.1 Mb) that were amplified in 7/8 cell lines, and in the 8q21 region there was a smaller 0.88 Mb region (86.62-87.50 Mb) amplified in 4/8 cell lines. A global expression analysis was performed for all these cell lines using a high-density oligonucleotide array to identify the genes whose expression correlated with amplification/deletion. Representative genes from 5 commonly amplified regions (REXO1L1, TMEM55A, TRMT12, MTSS1, EIF2C2, MYC) and 2 deleted regions (DPYSL2, NRG1 and NDRG1) were verified by qPCR and RT-qPCR for genomic and expression changes. Validation by RT-qPCR using RNA from 30 breast tumors showed that the TRMT12 (125.5 Mb) gene were overexpressed in many of the tumors. TRMT12 is homolog of a yeast gene encoding tRNA methyltransferase involved in biosythesis of a modified base in a tRNA. It would be interesting to explore the role of TRMT12 and the RNA processing pathway in tumorigenesis. Keywords: Comparative Genomic Hybridization (CGH), Breast cancer cell lines, Expression, BAC array, Oligo expression array

ORGANISM(S): Homo sapiens

PROVIDER: GSE6567 | GEO | 2007/05/07

SECONDARY ACCESSION(S): PRJNA98741

REPOSITORIES: GEO

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Project description:Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were identified. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ~359-kb and ~215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24-bp within interchromosomal paralogous LCRs of ~130-kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 5292 interchromosomal LCR substrate pairs, > 5-kb in size and sharing > 94% sequence identity that can potentially mediate chromosomal translocations. Additional proof for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55-bp in ~7.8-kb paralogous subunits of 95.3% sequence identity located in the ~579-kb (chr8) and ~287-kb (chr12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations throughout the human genome and provide a computationally determined genome-wide “recurrent translocation map”. Affymetrix Genome-Wide SNP Array 6.0 arrays (Affymetrix, Santa Clara, California) were employed to define the breakpoints on chromosomes 4, 8, 11, and 12. Analysis was performed according to the Genome-Wide Human SNP Nsp/Sty Assay Kit 5.0/6.0 protocol provided by the supplier. The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc.) and results were analyzed using Genotyping Console version 2.1 software. patient 1: Version 5 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 2: Version 5.1 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 3: The BAC emulated Version 6.1 OLIGO array was comprised of approximately 42,460 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). The 42.46K oligonucleotides (oligos) were selected from initial testing of 105,000 oligos derived from the Agilent eArray library with strict selection criteria and removal of repetitive sequences to ensure optimal performance with greater dynamic range. This targeted 42.46 K OLIGO array (V6 OLIGO) corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 5: The BAC emulated Version 6.3 OLIGO array was comprised of approximately 43,580 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 6: The BAC emulated Version 6.4 OLIGO array was comprised of approximately 43,700 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. Patient 4 was a known t(4;11) translocation patient form previous report and only ran on Affymetrix array.

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Chromosome 8 BAC Array CGH and Expression Studies Identify Amplification and Overexpression of TRMT12 in Breast Cancer

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