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Quality Control in array-CGH
We validated the quality of our custom-made CGH arrays by performing four sex-mismatched or sex-matched normal versus normal hybridizations.
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Fig. 1. BAC-based CGH array.A
normal versus normal control hybridizations on MCG Cancer Array-800; a representative genomic profile obtained from one of six normal versus normal control hybridizations. Clones are ordered from chromosome 1 to 22 and within each chromosome on the basis of the UCSC mapping position (http://genome.ucsc.edu/, version December 2000). Thresholds for copy number gain and loss are shown at within log2ratio of 0.2 and -0.2, respectively. |
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Fig. 1. BAC-based CGH array.B
Detection of hemizygous deletion on MCG Whole Genome Array-4500 in a patient with Williams syndrome (WMS: MIM194050). WMS is a contiguous gene syndrome that is characterized by a hemizygous deletion at chromosome 7q11.23. An array profile of chromosome 7 shows reduced fluorescent ratios at three BACs within the WMS critical region (arrow), indicating that our CGH array can discriminate one copy loss in a background of diploid genome. In the right panel, FISH confirmation clearly shows one copy loss of LIMK1 (green signal) within the WMS critical deletion. Red signals are for control BAC at 7q11.21. |
As shown in Fig.1, thresholds for copy number gain and loss are shown at log2ratio of 0.2 and -0.2, respectively. Under the condition, our custom-made MCG Array-4500, which harbors 4523 BACs through the entire whole genome, can discriminate one copy loss in a background of diploid genome. Haploinsufficiency of a specific gene(s) is well known to be causative of disease conditions in cancer28) as well as congenital disorders. Therefore, cryptic aberrations emerged from array-CGH analysis will make a beginning for identification of genes associated with cancer and unknown genetic diseases.
Various tumor suppressor genes have been identified from chromosomal regions involved in homozygous deletion. Indeed, representative tumor suppressors of p16 and PTEN were found in regions involved in homozygous deletions at bands 9p21 and 10q23, respectively. Therefore, novel homozyogous deletions detected at the submegabase level through CGH-array must be directly connected with identification of the place harboring gene(s) that acts as a tumor suppressor.
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Fig. 2. BAC-based CGH array in Cancer Genomics
MCG Cancer Array-800 profile from an esophageal squamous cell carcinoma cellline, KYSE410. Clones are ordered from chromosome 1 to 22 and within each chromosome on the basis of the UCSC mapping position (http://genome.ucsc.edu/, version December 2000). Thresholds for copy number gain and loss are shown at log2ratio of 0.2 and -0.2, respectively. Copy number amplification is detected on three BACs on 17q11.2, in which one BAC (RP11-62N23) harbors ERBB2. A homozygous deletion of the 9p21 region is also seen in this case. In the right panel, FISH reveals ERBB2 amplification on two markers chromosomes as HSRs (arrows). Red signals are detected as control at 17911.2 by BAC (RP11-229K15). |
As shown in Fig.2, our analysis of esophageal squamous cell carcinoma (ESC) cell line clearly demonstrates the usefulness of CGH array methodology for detection of homozygous deletions in tumors. In human genome, previous study have revealed many regions involved in loss of heterozygosity (LOH) and the possible existence of yet uncharacterized tumor suppressors within the loci. Precise mapping of those is often difficult or labor intensive by using existing technology. On the other hand, array-CGH can circumvent this difficulty, since high resolution array-CGH can discriminate submeganbase deletions.23) Indeed, recently, by using CGH-array we detected submicroscopic homozyogous deletion at 2q34 and could identify the Low density lipoprotein (LDL) receptor-related protein 1B gene ( LRP1B) as the most likely tumor suppressor in ESC.25) Subsequent genomic PCR showed frequent homozygous deletion of this gene in both ESC cell lines (6/44, 13.7%) and primary tumors (30/70, 42.9%). Moreover, LRP1B mRNA expression was frequently silenced in ESC lines even without its homozygous deletion (14/38, 36.8%). LRP1B-nonexpressing cells without its homozygous deletion were highly methylated in the LRP1B CpG island in both cell lines and primary tumors of ESC, and restoration of LRP1B expression in ESC cells reduced colony formation. Those results suggest that LRP1B may be involved in esophageal carcinogenesis through loss of function by homozygous deletion or transcriptional silencing by CpG island hypermethylation.25)
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